Treatment of rett syndrome using glycyl-L-2-methylprolyl-L-glutamic acid

ABSTRACT

This invention provides compounds, compositions and methods for treating Autism Spectrum Disorders (ASD) using glycyl-2-methylprolyl-glutamic acid (G-2-MePE) and analogs thereof. Autism Spectrum Disorders include Autism, Autistic Disorder, Asperger Syndrome, Childhood Disintegrative Disorder, Pervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS), Fragile X Syndrome, and Rett Syndrome. Compositions containing compounds include water-soluble formulations, water-in-oil micro-emulsions, water-in-oil coarse emulsions, water-in-oil liquid crystals, nanocapsules, tablets, and orally administered gels. The compounds and compositions of this invention can be administered intravenously, intraventricularly, parenterally, or orally, and can be effective in treating neurodegeneration, promoting neurological function, treating seizure activity and other symptoms of ASD, and can prolong life in animals including human beings having Autism Spectrum Disorders.

CLAIM OF PRIORITY

This application is a Continuation filed under 35 U.S.C. 120 of U.S.application Ser. No. 13/699,087, filed Jun. 5, 2013, which is a UnitedStates 371 National Phase application of PCT/US2012/000047, entitled“Treatment of Autism Spectrum Disorders UsingGlycyl-L-2-Methylprolyl-L-Glutamic Acid,” filed 27 Jan. 2012, whichclaims priority to U.S. Provisional Patent Application No. 61/462,141entitled “Rett Syndrome Therapy UsingGlycyl-2-Methylprolyl-L-Glutamate,” Larry Glass et al., inventors, filed27 Jan. 2011, and to U.S. Provisional Patent Application No. 61/492,248entitled “Treatment of Autism Spectrum Disorders UsingGlycyl-2-L-Methylprolyl-L-Glutamate,” Michael John Bickerdike et al.inventors, filed 1 Jun. 2011. Each of these applications is incorporatedherein fully by reference as if separately so incorporated.

FIELD OF THE INVENTION

This invention relates generally to therapy of Autism Spectrum Disorders(ASD), including autism, Fragile X Syndrome, Rett Syndrome (RTT),Autistic Disorder, Asperger Syndrome, Childhood Disintegrative Disorderand Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS),and Pathological Demand Avoidance (PDA). In particular, this inventionrelates to treatment of ASD using Glycyl-2-methyl-Prolyl-Glutamate(G-2-MePE).

BACKGROUND Description of Related Art

Autism Spectrum Disorders are becoming increasingly diagnosed. Autismspectrum disorders (ASD) are a collection of linked developmentaldisorders, characterized by abnormalities in social interaction andcommunication, restricted interests and repetitive behaviours. Currentclassification of ASD recognises five distinct forms: classical autismor Autistic Disorder, Asperger syndrome, Rett syndrome, childhooddisintegrative disorder and pervasive developmental disorder nototherwise specified (PDD-NOS). A sixth syndrome, pathological demandavoidance (PDA), is a further specific pervasive developmental disorder.

EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acidsGly-Pro-Glu) and its di-peptide derivatives Gly-Pro and Pro-Glu. EP 0366 638 discloses that GPE is effective as a neuromodulator and is ableto affect the electrical properties of neurons.

WO95/172904 discloses that GPE has neuroprotective properties and thatadministration of GPE can reduce damage to the central nervous system(CNS) by the prevention or inhibition of neuronal and glial cell death.

WO 98/14202 discloses that administration of GPE can increase theeffective amount of choline acetyltransferase (ChAT), glutamic aciddecarboxylase (GAD), and nitric oxide synthase (NOS) in the centralnervous system (CNS).

WO99/65509 discloses that increasing the effective amount of GPE in theCNS, such as by administration of GPE, can increase the effective amountof tyrosine hydroxylase (TH) in the CNS to increase TH-mediated dopamineproduction in the treatment of diseases such as Parkinson's disease.

WO02/16408 discloses certain GPE analogs having amino acid substitutionsand certain other modification that are capable of inducing aphysiological effect equivalent to GPE within a patient. Theapplications of the GPE analogs include the treatment of acute braininjury and neurodegenerative diseases, including injury or disease inthe CNS.

SUMMARY

There is no current, effective, treatment of ASD and patient care islimited to management of the symptoms.

This invention relates to synthetic analogs and peptidomimetics ofglycyl-L-prolyl-L-glutamic acid (GPE). In particular, this inventionrelates to GPE analogs and peptidomimetics that are anti-apoptotic andanti-necrotic, to methods of making them, to pharmaceutical compositionscontaining them, and to their use to enhance cognitive function and/ortreat memory disorders and to improve neuronal connectivity in animals.More specifically, this application relates to the methods of use of theGPE analog, G-2Methyl-Prolyl-Glutamic acid (G-2-MePE) in the treatmentof ASD.

The U.S. Pat. No. 7,041,314 discloses compositions of matter and methodsof use of G-2-MePE

In one aspect, this invention provides compounds of Formula 1 andFormula 2:

where m is 0 or 1;n is 0 or 1;X is H or —NR⁶R⁷;Y is H, alkyl, —CO₂R⁵, or —CONR⁶R⁷;Z is H, alkyl, —CO₂R⁵ or —CONR⁶R⁷;R¹ is H, alkyl, or aralkyl;R², R³, and R⁴ are independently H or alkyl;each R⁵ is independently H, alkyl, or a fatty alcohol residue;each R⁶ and R⁷ is independently H, alkyl, or aralkyl, or —NR⁶R⁷ ispyrrolidino, piperidino, or morpholino;or a lactone formed when a compound where Y is —CO₂(alkyl) and Z is—CO₂H or where Y is —CO₂H and Z is —CO₂(alkyl) is lactonized;and the pharmaceutically acceptable salts thereof,provided that the compound is not GPE, N-Me-GPE, GPE amide, APE, GPQ ora salt thereof.

Another aspect the invention provides methods for treatment of an animalhaving a Autism Spectrum Disorder comprising administration of aneffective amount of Glycyl-L-2-Methylprolyl-L-Glutamic Acid (G-2-MePE)to the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

Color Drawings

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

This invention is described with reference to specific embodimentsthereof. Other aspects and features of this invention can be understoodwith reference to the Figures, in which:

FIG. 1 is a general scheme for preparation of synthetic analogs of GPEof the invention.

FIGS. 2 and 3 depict schemes for modifying glycine residues on GPE.

FIGS. 4 through 9 depict schemes for modifying glutamic acid residues ofGPE.

FIGS. 10 and 11 depict schemes for modifying peptide linkages of GPE.

FIGS. 12-15 depict graphs summarizing results of testing neurons invitro with GPE or G-2-MePE and okadaic acid.

FIG. 12 depicts a graph showing effects of GPE on cortical neuronsinjured with okadaic acid.

FIG. 13 depicts a graph showing effects of G-2-MePE on cortical neuronsinjured with okadaic acid.

FIG. 14 depicts a graph showing effects of G-2-MePE, GPE on cerebellarmicroexplants injured with okadaic acid.

FIG. 15 depicts a graph showing effects of G-2-MePE or GPE on striatalcells injured with okadaic acid.

FIG. 16 shows the effects of subcutaneous injection of G-2-MePE (atdoses of 0.012, 0.12, 1.2 and 12 mg/kg) on the number of ChAT-positiveneurons in the striatum of 18-month old rats.

FIG. 17 shows effects of G-2-MePE treatment on spatial memory retentionin middle-aged 12-month old rats.

FIGS. 18A and 18B show effects of G-2-MePE on spatial working memory ofaged (17-month old) rats in an 8-arm radial maze following 3-weeks oftreatment and a nine day washout.

FIG. 18A shows the maze acquisition profiles across days for thedifferent groups. FIG. 18B shows the proportion of correct maze choicesaveraged across days for the groups.

FIG. 19A shows effects of a single intraperitoneal administration of 4doses of G-2-MePE on neuroblast proliferation as assessed by the numberof PCNA positive cells in the subventricular zone (SVZ) of aged rats.

FIG. 19B shows effects of a single intraperitoneal administration of 4doses of G-2-MePE on co-localisation of PCNA and doublecortin stainingin a rat treated with the highest dose of G-2-MePE (right panel)compared to the vehicle treated rat (left panel).

FIG. 19C shows effects of G-2-MePE on neuroblast proliferation asassessed by PCNA immunohistochemical staining in middle-aged rats.

FIG. 20A shows a significant increase in the number of reactiveastrocytes as assessed by GFAP staining in the hippocampus in aged ratscompared to young rats (*p<0.01) and middle aged rats (*p<0.01).

FIG. 20B shows a photograph of a section of cerebral cortex of an agedrat, showing astrocytes as assessed with GFAP staining, some of whichare associated with formation of capillaries (arrows).

FIG. 20C shows dose-dependent effects of G-2-MePE treatment (at doses of0.12, 0.12, 1.2 and 12 mg/kg/day) on reduction of the number ofastrocytes as assayed using GFAP staining in the CA4 sub-region of thehippocampus in aged rats.

FIG. 20D shows dose-dependent effects of G-2-MePE treatment (at doses of0.12, 0.12, 1.2 and 12 mg/kg/day) on reduction of the number ofastrocytes as assayed using GFAP staining in the cerebellar cortex.

FIG. 21 shows pharmacokinetic properties of GPE and G-2-MePE in thecirculation of rats after intravenous injection.

FIG. 22 shows the effects of G-2-MePE on increased survival duration inMeCP2 deficient mice compared to saline-treated MeCP2 deficient mice.

FIG. 23 shows the effects of G-2-MePE on the hippocampal long-termpotentiation as measured by the fEPSP slope in MeCP2 deficient mice,compared to saline-treated MeCP2 deficient mice.

FIG. 24 depicts a graph showing effects of G-2-MePE on dendrite lengthas a function of distance from the cell soma.

DETAILED DESCRIPTION Definitions

The term “about” with reference to a dosage or time refers to aparticular variable and a range around that variable that is withinnormal measurement error is within 20% of the value of the variable. Theterm “about” with reference to a result observed means the variation iswithin 20% of the value of the observed variable.

The term “alkyl” means a linear saturated hydrocarbyl group having fromone to six carbon atoms, or a branched or cyclic saturated hydrocarbylgroup having from three to six carbon atoms. Exemplary alkyl groupsinclude straight and branched chain, or cyclic alkyl groups, methyl,ethyl, isopropyl, cyclopropyl, tert-butyl, cyclopropylmethyl, and hexyl.

The term “animal” includes humans and non-human animals, such asdomestic animals (cats, dogs, and the like) and farm animals (cattle,horses, sheep, goats, swine, and the like).

The term “aralkyl” means a group of the formula —(CH₂)₁₋₂Ar, where Ar isa 5- or 6-membered carbocyclic or heterocyclic aromatic ring, optionallysubstituted with 1 to 3 substituents selected from Cl, Br, —OH,—O-alkyl, —CO₂R⁸ (where R⁸ is H or alkyl), or —NR⁸R⁹, where R⁸ is asdescribed previously and R⁹ is H or alkyl. Exemplary aralkyl groupsinclude benzyl, 2-chlorobenzyl, 4-(dimethylamino)benzyl, phenethyl,1-pyrrolylmethyl, 2-thienylmethyl, and 3-pyridylmethyl.

The term “disease” includes any unhealthy condition of an animalincluding particularly Parkinson's disease, Huntington's disease,Alzheimer's disease, multiple sclerosis, diabetes, motor disorders,seizures, cognitive dysfunctions due to aging and Autism SpectrumDisorders including autism, Fragile X Syndrome, Rett Syndrome (RTT),Autistic Disorder, Asperger Syndrome, Childhood Disintegrative Disorderand Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS),and Pathological Demand Avoidance (PDA).

The term “fatty alcohol residue” is a linear hydrocarbyl group havingfrom seven to twenty carbon atoms, optionally containing up to threecarbon-carbon double bonds. Exemplary fatty alcohol residues includedecyl, pentadecyl, hexadecyl (cetyl), octadecyl (stearyl), oleyl,linoleyl, and eicosyl.

The term “growth factor” means an extracellular polypeptide-signalingmolecule that stimulates a cell to grow or proliferate.

The term “injury” includes any acute damage of an animal includingnon-hemorrhagic stroke, traumatic brain injury, perinatal asphyxiaassociated with fetal distress such as that following abruption, cordocclusion or associated with intrauterine growth retardation, perinatalasphyxia associated with failure of adequate resuscitation orrespiration, severe CNS insults associated with near miss drowning, nearmiss cot death, carbon monoxide inhalation, ammonia or other gaseousintoxication, cardiac arrest, coma, meningitis, hypoglycemia and statusepilepticus, episodes of cerebral asphyxia associated with coronarybypass surgery, hypotensive episodes and hypertensive crises, cerebraltrauma and toxic injury.

“Memory disorders” or “cognitive disorders” are disorders characterizedby permanent or temporary impairment or loss of ability to learn,memorize or recall information. Memory disorder can result from normalaging, injury to the brain, tumors, neurodegenerative disease, vascularconditions, genetic conditions (Huntington's disease), hydrocephalus,other diseases (Pick's disease, Creutzfeld-Jakob disease, AIDS,meningitis), toxic substances, nutritional deficiency, biochemicaldisorders, psychological or psychiatric dysfunctions. The presence ofmemory disorder in a human can be established thorough examination ofpatient history, physical examination, laboratory tests, imagining testsand neuropsychological tests. Standard neuropsychological tests includebut are not limited to Brief Visual Memory Test-Revised (BVMT-R),Cambridge Neuropsychological Test Automated Battery (CANTAB), Children'sMemory Scale (CMS), Contextual Memory Test, Continuous RecognitionMemory Test (CMRT), Controlled Oral Word Association Test and MemoryFunctioning Questionnaire, Denman Neuropsychology Memory Scale, DigitSpan and Letter Number Sequence sub-test of the Wechsler AdultIntelligence Scale-III, Fuld Object Memory Evaluation (FOME),Graham-Kendall Memory for Designs Test, Guild Memory Test, HopkinsVerbal Learning Test, Learning and Memory Battery (LAMB), MemoryAssessment Clinic Self-Rating Scale (MAC-S), Memory Assessment Scales(MAS), Randt Memory Test, Recognition memory Test (RMT), Rey Auditoryand Verbal Learning Test (RAVLT), Rivermead Behavioral Memory Test,Russell's Version of the Wechsler Memory Scale (RWMS), Spatial WorkingMemory, Test of Memory and Learning (TOMAL), Vermont Memory Scale (VMS),Wechsler Memory Scale, Wide Range Assessment of Memory and Learning(WRAML).

The term “pharmaceutically acceptable excipient” means an excipient thatis useful in preparing a pharmaceutical composition that is generallysafe, non-toxic, and desirable, and includes excipients that areacceptable for veterinary use as well as for human pharmaceutical use.Such excipients may be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

The term “pharmaceutically acceptable salt” means a salt that ispharmaceutically acceptable and has the desired pharmacologicalproperties. Such salts include salts that can be formed where acidicprotons present in the compounds react with inorganic or organic bases.Suitable inorganic salts include those formed with the alkali metals,e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitableorganic salts include those formed with organic bases such as aminese.g. ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Salts also include acid addition saltsformed by reaction of an amine group or groups present in the compoundwith an acid. Suitable acids include inorganic acids (e.g. hydrochloricand hydrobromic acids) and organic acids (e.g. acetic acid, citric acid,maleic acid, and alkane- and arene-sulfonic acids such asmethanesulfonic acid and benzenesulfonic acid). When there are twoacidic groups present in a compound, a pharmaceutically acceptable saltmay be a mono-acid mono-salt or a di-salt; and similarly where there aremore than two acidic groups present, some or all of such groups can besalified. The same reasoning can be applied when two or more aminegroups are present in a compound.

The term “protecting group” is a group that selectively blocks one ormore reactive sites in a multifunctional compound such that a chemicalreaction can be carried out selectively on another unprotected reactivesite and such that the group can readily be removed after the selectivereaction is complete.

The term “therapeutically effective amount” means the amount of an agentthat, when administered to an animal for treating a disease, issufficient to effect treatment for that disease as measured using a testsystem recognized in the art.

The term “treating” or “treatment” of a disease may include preventingthe disease from occurring in an animal that may be predisposed to thedisease but does not yet experience or exhibit symptoms of the disease(prophylactic treatment), inhibiting the disease (slowing or arrestingits development), providing relief from the symptoms or side-effects ofthe disease (including palliative treatment), and relieving the disease(causing regression of the disease).

The term “functional deficit” means a behavioral deficit associated withneurological damage. Such deficits include deficits of gait, as observedin patients with Parkinson's disease, motor abnormalities as observed inpatients with Huntington's disease. Functional deficit also includesabnormal foot placement and memory disorders described herein.

The terms “G-2-MePE” and “NNZ-2566” meansL-Glycyl-2-methyl-L-Prolyl-L-Glutamate.

The term “seizure” means an abnormal pattern of neural activity in thebrain that results in a motor deficit or lack of motor control resultingin abnormal motion, including spasmodic motion. “Seizure” includeselectroencephalographic abnormalities, whether or not accompanied byabnormal motor activity.

Implicit hydrogen atoms (such as hydrogen atoms on a pyrrolidine ring,etc.) are omitted from the formulae for clarity, but should beunderstood to be present.

Autism Spectrum Disorders

Autism spectrum disorders (ASDs) are a collection of linkeddevelopmental disorders, characterized by abnormalities in socialinteraction and communication, restricted interests and repetitivebehaviours. Current classification of ASDs recognises five distinctforms: classical autism or Autistic Disorder, Asperger syndrome, Rettsyndrome, childhood disintegrative disorder and pervasive developmentaldisorder not otherwise specified (PDD-NOS). A sixth syndrome,pathological demand avoidance (PDA), is a further specific pervasivedevelopmental disorder. However, while PDA is increasingly recognised asan ASD, it is not yet part of the Diagnostic and Statistical Manual ofMental Disorders (DSM-IV), published by the American PsychiatricAssociation, nor is it part of the proposed revision, the DSM-V.

Autism

Classical autism is a highly variable neurodevelopmental disorder. It istypically diagnosed during infancy or early childhood, with overtsymptoms often apparent from the age of 6 months, and becomingestablished by 2-3 years. According to the criteria set out in theDSM-IV, diagnosis of autism requires a triad of symptoms to be present,including (a) impairments in social interaction, (b) impairments incommunication and (c) restricted and repetitive interests andbehaviours. Other dysfunctions, such as atypical eating, are also commonbut are not essential for diagnosis. Of these impairments, socialinteraction impairments are particularly important for diagnosis, andtwo of the following impairments must be present for a diagnosis ofautism:

-   -   (i) impairments in the use of multiple nonverbal behaviors (e.g.        eye contact) to regulate social interaction;    -   (ii) failure to develop peer relationships appropriate to        developmental level;    -   (iii) lack of spontaneous seeking to share enjoyment, interests,        or achievements;    -   (iv) lack of social or emotional reciprocity.

Communication impairments in autism may be manifest in one or more ofthe following ways: delay in (or total lack of) the development ofspoken language; marked impairment in the ability to initiate or sustaina conversation; stereotyped and repetitive use of language; and/or alack of spontaneous make-believe play. Restricted, repetitive andstereotyped patterns of behavior is also required for diagnosis, such aspreoccupation with one or more interest considered abnormal inintensity, inflexible adherence to routines or rituals, repetitive motormannerisms and/or persistent focus on parts of objects.

Lastly, for a diagnosis of autism, it is necessary that the impairmentin the functioning of at least one area (i.e. social interaction,language, or imaginative play) should have an onset at less than 3 yearsof age.

Autism is commonly associated with epilepsy or epileptiform activity inthe electroencephalogram (EEG). As many as 60 percent of patients withautism have epileptiform activity in their EEGs (Spence and Schneider,2009 Ped Res 65: 599-606).

Autism is also associated with disturbances in function of IGF-1, whichis depleted in the Central Nervous System (CNS) in patients with autism(Riikonen et al., 2006 Devel Med Child Neurol 48: 751-755). IGF-1 levelsin the CNS increase in patients with autism after treatment with agentsthat reduce symptoms such as fluoxetine (Makkonen et al., 2011Neuropediatrics 42:207-209).

Importantly, autism shares features of Rett Syndrome and Fragile XSyndrome in relation to neuronal connectivity. All three disorders arecharacterised by defects in synaptic function and neuronal connectivity.This is reflected in studies of post mortem human brain in these patientgroups, which all show failure to form normal synaptic connections. Thisis reflected in altered morphological characteristics, being either areduction in neuron dendritic spine density, or enhanced dendritic spinedensity but associated with immature synapses. This is reflected inanimal models of autism, Rett Syndrome and Fragile X Syndrome, which arebased on genetic changes known to be pathological in these disorders. Inthese animal models, neuronal connectivity defects are revealedmorphologically, and also as a failure of Long Term Potentiation (LTP).This is important since IGF-1, IGF-1[1-3] and G-2-MePE increase synapseformation.

Asperger Syndrome

Asperger syndrome or Asperger Disorder is similar to autism, and sharescertain features. Like autism, Asperger syndrome is also characterizedby impairment in social interaction and this is accompanied byrestricted and repetitive interests and behavior. Thus, diagnosis ofAsperger syndrome is characterized by the same triad of impairments asautism. However, it differs from the other ASDs by having no generaldelay in language or cognitive development and no deficit in interest inthe subject's environment. Moreover, Asperger syndrome is typically lesssevere in symptomology than classical autism and Asperger's patients mayfunction with self-sufficiency and lead relatively normal lives.

Childhood Disintegrative Disorder

Childhood disintegrative disorder (CDD), also known as Heller syndrome,is a condition in which children develop normally until age 2-4 years(i.e. later than in Autism and Rett syndrome), but then demonstrate asevere loss of social, communication and other skills. Childhooddisintegrative disorder is very much like autism and both involve normaldevelopment followed by significant loss of language, social play andmotor skills. However, childhood disintegrative disorder typicallyoccurs later than autism, involves a more dramatic loss of skills and isfar less common.

Diagnosis of CDD is dependent on dramatic loss of previously acquiredskills in two or more of the following areas: language, social skills,play, motor skills (such as a dramatic decline in the ability to walk,climb, grasp, etc), bowel or bladder control (despite previously beingtoilet-trained). The loss of developmental skills may be abrupt and takeplace over the course of days to weeks or may be more gradual.

Pervasive Developmental Disorder—not Otherwise Specified (PDD-NOS)

Pervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS) is anASD that describes patients exhibiting some, but not all, of thesymptoms associated with other well defined ASDs. The key criteria fordiagnosis of an ASD include difficulty socializing with others,repetitive behaviors, and heightened sensitivities to certain stimuli.These are all found in the ASDs described above. However, autism,Asperger syndrome, Rett syndrome and childhood disintegrative disorderall have other features that enable their specific diagnosis. Whenspecific diagnosis of one of these four disorders cannot be made, butASD is apparent, a diagnosis of PDD-NOS is made. Such a diagnosis mayresult from symptoms starting at a later age than is applicable forother conditions in the spectrum.

Rett Syndrome

Rett Syndrome (RTT) is a neurodevelopmental disorder that almostexclusively affects females (1 in 10:000 live births). RTT is classifiedas an autism spectrum disorder (Diagnostic and Statistical Manual ofMental Disorders, Fourth Edition-Revised (DSM-IV-R). Approximately16,000 patients are currently affected by it in the U.S.A. (RettSyndrome Research Trust data). For a diagnosis of Rett syndrome, thefollowing symptoms are characteristic: impaired development from age6-18 months; slowing of the rate of head growth starting from betweenage 3 months and 4 years; severely impaired language; repetitive andstereotypic hand movements; and gait abnormalities, e.g. toe-walking orunsteady stiff-legged walk. There are in addition, a number ofsupportive criteria that may help diagnosis of Rett Syndrome, but arenot essential for a diagnosis. These include breathing difficulties, EEGabnormalities, seizures, muscle rigidity and spasticity, scoliosis(curving of the spine), teeth-grinding, small hands and feet in relationto height, growth retardation, decreased body fat and muscle mass,abnormal sleep patterns, irritability or agitation, chewing and/orswallowing difficulties, poor circulation and constipation.

The onset of RTT usually begins between 6-18 months of age with aslowing of development and growth rates. This is followed by aregression phase (typically in children aged 1-4 years of age),pseudo-stationary phase (2-10 years of age) and a subsequent progressivelate motor deterioration state. RTT symptoms include sudden decelerationof growth and regression in language and motor skills includingpurposeful hand movements being replaced by stereotypical movements,autistic features, panic-like attacks, sleep cycle disturbances,tremors, seizures, respiratory dysfunctions (episodic apnea, hyperpnea),apraxia, dystonia, dyskinesia, hypotonia, progressive kyphosis orscoliosis and severe cognitive impairment. Most RTT patients surviveinto adulthood with severe disabilities and require 24-hour-a-day care.

Between 85% and 95% cases of RTT are reported to be caused by a mutationof the Mecp2 gene (Amir et al. 1999. Nat Genet 23:185-188; Rett SyndromeResearch Trust)—a gene encoding methyl-CpG-binding protein 2 (MeCP2).Mecp2 maps to the X-chromosome (location Xq28) and for this reason,mutations to the gene in males are usually lethal. While RTT is agenetic disorder, less than 1% of recorded cases are inherited; almostall mutations of Mecp2 occur de novo, with two thirds caused bymutations at 8 CpG dinucleotides (R106, R133, T158, R168, R255, R270,R294 and R306) located on the third and fourth exons.

MeCP2 is a protein that binds methylated CpG dinucleotides to exerttranscriptional silencing of DNA in the CNS. The key effect of areduction or absence of MeCP2 appears to be an impairment of dendriticspine development and the formation of synapses. MeCP2 expressionappears to temporally correlate with brain maturation, explaining whysymptoms typically appear around 18 months of age.

Presenting Features Common to ASDs

Taking the ASDs together, it is clear that there are commonalities inpresenting symptoms among all 5 forms. These common features areimpairments in normal social competences, and repetitive behaviours. Inall but Asperger syndrome there is also a consistent presentation ofdelayed intellectual development most commonly manifest as a shortfallin language skills. Cognitive loss relative to normal parameters for theage is often quite marked in autism, Rett Syndrome, CDD and PDD-NOS. Thepresence of epilepsy or abnormal activity in the EEG is also common toautism, Fragile X Syndrome and Rett Syndrome. Epilepsy arises insituations of abnormal neuronal connectivity, Impaired neuronalconnectivity and deranged synaptic function is a common feature ofautism, Fragile X Syndrome and Rett Syndrome and of animal models ofthese conditions.

Genetic Models of ASD

To offer validity, animal models of ASDs must demonstrate similarsymptoms to the clinical conditions and have a reasonable degree of facevalidity regarding the etiology of those symptoms. It is known thatclassical autism may be caused by many different genetic impairments andno single genetic defect is thought to account for more than a fewpercent of autism cases. Indeed, recent studies have revealed numerousde novo structural variations of chromosome locations thought tounderlie ASD, in addition to rare inherited genetic defects (Marshall etal, 2008; Sebat et al, 2007). Thus, copy number variation (CNV),translocation and inversion of gene sequences at 20 key sites or more,including 1p, 5q, 7q, 15q, 16p, 17p and Xq, have been mapped as ASDloci.

However, despite the polygenetic background underlying ASD, and thecomplexity of the etiology, it is known that certain genetic defects canproduce ASD. Some of the best characterized defects arise fromchromosomal aberrations of genes that code for a cluster of postsynapticdensity proteins, including neuroligin-3 (NLGN3), neuroligin-4 (NLGN4),neurexin-1α (NRXN1) and shank3 (Sebat et al, 2007). Importantly, thesedefects point to altered synaptic function and therefore disturbedneuronal connectivity as a final common pathway in autism and relateddisorders (Minshew and Williams 2007 Arch Neurol. 64:945-950; Gilman etal., 2011 Neuron. 70:898-907). Such connectivity deficits are reflectedin morphological findings in post mortem examination, which revealincreased dendritic spine density in autism (Hutsler and Zhang 2010Brain Res. 1309:83-94).

NLGN3 and NLGN4 are postsynaptic cell-adhesion molecules present inglutamatergic synapses. They play a role in coordinating presynapticcontact to the postsynaptic site and also interact with the postsynapticscaffolding protein shank3. Mutations to NLGN3 and NLGN4 have beenobserved in the ASD population and account for perhaps 1% of all ASDcases (Lintas & Persico, 2008). Jamain and colleagues first reported amissense to NLGN3 and a frameshift to NLGN4 in two unrelated subjects,resulting in Asperger syndrome and classical autism respectively (Jamainet al, 2003). While the incidence of NLGN3 or NLGN4 mutations in the ASDpopulation is low (indeed, no much mutations were observed in a study of96 ASD patients in a Canadian study; Gauthier et al, 2005), it has beenconfirmed in preclinical studies that neuroligin mutations can indeedproduce of model of autistic symptoms. Thus, introduction to mice of thesame R451C missense to NLGN3 that has been reported clinically resultsin a mutant mouse strain showing reduced social interaction and enhancedinhibitory synaptic transmission (Tabuchi et al, 2007).

The R451C mutant therefore mouse represents a model for ASD based uponNLGN3 mutation. In this case, mutation at the R451 position of NLGN3results in a ‘gain-of-function’ mutation.

In contrast, modeling the clinical mutation of NLGN4 in mice is achievedby a ‘loss-of-function’ mutation of NLGN4 (a classical knockout model).In this model, mutant mice display a social interaction deficit andreduced ultrasonic vocalization (Jamain et al, 2008). Communicationdeficits are central to clinical ASDs and in the NLGN4 knockout mice areduction in ultrasonic vocalizations from male mice exposed towild-type female counterparts supports the face validity of the strainas a model of ASD.

Presynaptic neurexin proteins induce postsynaptic differentiation inopposing dendrites through interactions with postsynaptic neuroligincounterparts. Mutations of the neurexin-1α (NRXN1) gene have beenreported in numerous studies (Sebat et al, 2007; Marshall et al, 2008;Kim et al, 2008; Yan et al, 2008) and these have been observed in theform of copy-number variants. As with NLGN mutations, when a mutation ofthe NRXN1 gene is introduced to mice (in the form of gene knockout), amutant strain with certain ASD-like features is produced (Etherton etal, 2009). These NRXN1 knockout mice show a decrease in hippocampalminiature excitatory postsynaptic current (mEPSC) frequency and adecreased input-output relationship of evoked currents. Theseelectrophysiological effects relate to decreased excitatory transmissionin the hippocampus. In addition to decreased excitatoryneurotransmission, NRXN1 knockout mice exhibit a decrease in pre-pulseinhibition, though social behaviour appears, to be unaffected (Ethertonet al, 2009).

Sharing certain features with the neurexin-NLGN trans-synapticconstruct, cell adhesion molecule 1 (CADM1) is an immunoglobulin familyprotein present both pre- and post-synaptically that is also involved insynaptic trans-cell adhesion activity (Biederer et al, 2002). Mutationsto the CADM1 gene have been detected in ASD patients and appear torepresent a further possible cause of these conditions (Zhiling et al,2008).

Analysis of CADM1 knockout mice reveals that these animals showincreased anxiety-related behavior, impaired social interaction andimpaired social memory and recognition. In addition CADM1 knockout micedemonstrate poorer motor skills (Takayanagi et al, 2010). Thesedysfunctions are again consistent with ASD symptomatology.

22q13 deletion syndrome (also known as Phelan-McDermid Syndrome), is arare genetic disorder caused by a microdeletion at the q13.3 terminalend of chromosome 22. This microdeletion is rarely uncovered by typicalgenetic screening and a fluorescence in situ hybridization test isrecommended to confirm the diagnosis. Recent work indicates the syndromeis caused by errors in the gene shank3, a postsynaptic density proteincritical for normal neuronal functioning. Interestingly, errors in thisgene have also been associated with ASD and 22q13 deletion syndrome cancommonly lead to an ASD diagnosis (Durand et al, 2007; Moessner et al,2007; Sykes et al, 2009). Given the close association of 22q13 deletionsyndrome and the consequential diagnosis of ASD, a mutant mouse model ofthis mutation has been developed.

The shank3 knockout mouse exhibits several deficits that mirror ASDsymptoms, including reduced ultrasonic vocalizations (i.e. diminishedsocial communication) as well as impaired social interaction timebetween mice. In addition, these mice have impaired hippocampal CA1excitatory transmission, measured by input-output relationship of evokedcurrents and impaired long-term potentiation (LTP). LTP is believed tobe a physiological process underlying memory formation andconsolidation. Thus, the model exhibits a similar phenotype to the NLGN4knockout, consistent with ASD.

As has been noted, 22q13 deletion syndrome itself is very rare. However,it provides important information that involvement specific genes mayhave in the etiology of ASDs. In addition to shank3, this disorderreveals a further possible gene defect in ASD. Of the 50 or so cases of22q1.3 deletion syndrome described, all but one have a gene deletionthat extends beyond shank3 to include a further gene, known as the IsletBrain-2 gene (IB2) (Sebat et al, 2007). The IB2 protein interacts withmany other proteins including MAP kinases and amyloid precursor protein,appears to influence protein trafficking in neurites, and is enriched atpostsynaptic densities (Giza et al, 2010). Mice lacking the protein(IB2−/− knockout mice) exhibit impaired social interaction (reducedsocial sniffing and interaction time), reduced exploration and cognitiveand motoric deficits (Giza et al, 2010). This behavioral phenotype wasassociated with reduced excitatory transmission in cerebellar cells. Aswith shank3 knockout, the phenotype of IB2 mutation is therefore alsoconsistent with ASD.

In addition to the animal models of postsynaptic density protein defectsdescribed above, other monogenetic syndromes that share various featureswith ASDs can lead to autism offer another avenue for drug targeting ofASD. An excellent example of this is Fragile X Syndrome.

Fragile X Syndrome (FXS) is caused by the expansion of a singletrinucleotide gene sequence (CGG) on the X-chromosome that results infailure to express the protein coded by the fmr1 gene. FMR1 (fragile Xmental retardation 1) is a protein required for normal neuraldevelopment. FXS can cause a child to have autism (Hagerman et al,2010); in 2-6% of all children diagnosed with autism the cause is FXSgene mutation. Moreover, approximately 30% of FXS children have somedegree of autism and a further 30% are diagnosed with PDD-NOS (Hagermanet al, 2010). Indeed, Fragile X Syndrome is the most common known singlegene cause of autism. FMR1 knockout mice have been developed as a modelof FXS and, therefore, as a further model of ASD. Knockout mutation ofthe FMR1 gene has been shown to result in neuronal connectivity deficitssuch as abnormal dendritic spine development and pruning (Comery et al,1997), along with an associated dysregulation of dendritic scaffoldproteins (including shank1) and glutamate receptor subunits inpostsynaptic densities (Schütt et al, 2009). These effects on dendritemorphology results deficits in functional measures of connectivity suchas impaired LTP in the cortex and amygdala (Zhao et al, 2005) andhippocampus (Lauterborn et al, 2007), as well as impaired cognition(Kreuger et al, 2011) and an enhancement in social anxiety (Spencer etal, 2005). These connectivity deficits are mirrored in FXS patients, whoshow enhanced dendritic spine density in post mortem analyses (Irwin etal., 2000 Cereb Cortex 10:1034-1048). This enhanced dendritic spinedensity is accompanied by immature synapses (Klemmer et al., 2011 J BiolChem. 286:25495-25504), i.e. may represent a functionally immaturestate.

In contrast to the ASDs of autism, Asperger, CDD and PDD-NOS, Rettsyndrome appears to have an almost monogenetic basis and may be modeledin mice with good face validity. Rett syndrome is thought be caused, inup to 96% of cases, by a defect in the Mecp2 gene (Zoghbi, 2005). As aresult, MeCP2 knockout mutant mice provide an animal model with all thehallmarks of clinical Rett syndrome, with a phenotype showing someoverlap with the NLGN4, shank3 and IB2 knockout models of ASD. Thus,MeCP2 knockout mice display a clear impairment in LTP in the hippocampusalong with a corresponding decrease in social and spatial memory(Moretti et al, 2006) and impaired object recognition (Schaevitz et al,2010). This impairment in LTP is accompanied by a decrease in dendriticspine density. Patients with Rett Syndrome show reduced dendritic spinedensity (Belichenko et al., 1994 Neuroreport 5:1509-1513).

Thus, ASDs in human beings share many features of cognitive ordevelopmental disorders in animals, including rodents. Therefore,studies of therapies of ASDs in rodents such as mice and rats arereasonably predictive of results obtained in human beings. A commonfeature seen in autism, Fragile X Syndrome and Rett Syndrome is thepresence of neuronal connectivity deficits, reflected in eitherdecreased dendritic spine density or enhanced dendritic spine densitywith immature synapses. The functional consequences of thesemorphological changes are similar in animal models of these disorders,reflected as deficits in LTP, for example.

Treatment of Clinical ASD and ASD Animal Models with G-2-MePE

As described above, a conserved pathology is observed in ASDs thatcomprises impaired neurite development, impaired synaptic connectivityand a corresponding impairment in social and cognitive functioning as aresult. Such synaptic dysfunctions result from genetically alteredfunctions of postsynaptic density proteins. Normal neurite growth andpostsynaptic development may be regulated and augmented by growthfactors such as brain derived neurotrophic factor (BDNF; Chapleau et al,2009) and insulin-like growth factor-1 (IGF-1; Riikonen et al, 2006;Tropea et al, 2009). Indeed, IGF-1 is essential for normal dendriticspine growth and synapse formation (Cheng et al., 2003 J Neurosci Res.73:1-9), Drugs that promote growth factor function are therefore of usein the treatment of progressive developmental disorders such as ASDs.G-2-MePE is a small molecule methylated analog of the terminaltripeptide of IGF-1, IGF1(1-3). As an IGF-1 mimetic analog, G-2-MePEexerts trophic and neuroprotective effects in various animal models.G-2-MePE is therefore effective at treating ASD symptoms such as thoserelating to synaptic dysfunctions resulting from the gene mutationsdescribed above.

In clinical terms, ASD patients, presenting with autism, Aspergersyndrome, Rett syndrome, childhood disintegrative disorder and PDD-NOS,as well as patients with 22q13 deletion syndrome, Fragile X Syndrome andpathological demand avoidance are treated with G-2-MePE. Patientsexhibit social and communication impairments as well as cognitivedeficit. Treatment with G-2-MePE, for example, on a daily basis and inanother example, by the oral route, is observed to induce an improvementin stereotypic repetitive movements, improved social functioning andimproved cognitive performance following drug treatment.

In animal models of ASDs, daily G-2-MePE treatment by oral gavage orintraperitoneal injection to knockout mice will improve ASD-likesymptoms. G-2-MePE is effective in the following ASD mutant mousemodels: NLGN3 (R451C) mutant, NLGN4 knockout, NRXN1 knockout, CADM1knockout, shank3 knockout, IB2 knockout, FMR1 knockout and MeCP2knockout. When administered sub-chronically (1-10 weeks) on a dailybasis, G-2-MePE is effective at improving LTP in the hippocampusfollowing burst stimulation or high frequency stimulation. Similarly,G-2-MePE increases excitatory neurotransmission as measured by fieldextracellular postsynaptic potential electrophysiological recordings incortex, hippocampus and cerebellum. As a result of improved excitatoryneurotransmission (reversal of observed ASD-like neurotransmissiondeficit), G-2-MePE is observed to improve cognitive and motoric outcometests of cognitive performance. Thus, G-2-MePE improves performance inthe Morris water maze and radial arm maze tests. In models of socialinteraction, G-2-MePE, administered to ASD mutant mice, increases timespent by knockout males in social interaction with wild-type females. Inaddition, ultrasonic vocalizations to female wild type mice isincreased. In models in which longevity is observed to be reduced inmutant mice compared to wild-type controls (such as the MeCP2 knockoutmouse model of Rett Syndrome), treatment with G-2-MePE increases thelifespan of the animals.

G-2-MePE has been found to inhibit non-convulsive seizures (NCS) inanimals with hypoxic-ischemic injuries caused by middle cerebral arteryocclusion (MCaO; U.S. Pat. No. 7,714,020; Lu et al., NNZ-2566, aglypromate analog, attenuates brain ischemia-induced nonconvulsiveseizures in rats, J Cerebral Blood Flow metabolism (2009) 1-9) andinhibits neuroinflammation in animals with penetrating ballistic injury(pTBI; Wei et al., NNZ-2566 treatment inhibits neuroinflammation andpro-inflammatory cytokine expression induced by experimental penetratingballistic-like brain injury in rats, J. Neuroinflammation (2009) 6:19,1-10).

Our findings that G-2-MePE also are effective in treating Rett Syndromeand ASDs, are completely unexpected based on the prior art. This isbecause the NCS in the MCaO model is caused by hypoxia-ischemia and theinflammatory cytokine expression in the pTBI model is caused bypenetrating trauma, both of which are acute insults that are verydifferent from the chronic effects of MECP2 or other mutations onsynaptic maturation.

Because G-2-MePE is a member of the compounds of GPE analogs disclosedherein, any of the disclosed compounds also can be effective in treatingsymptoms of ASDs. Further, because compounds and methods of thisinvention address underlying neurological mechanisms (e.g., decreaseneural inflammation by inhibiting release of inflammatory cytokines),this invention can provide more than short-term management of symptoms.Rather, compounds and methods of this invention can improve neuralfunction, promote neuronal cell migration, promote neurogenesis, promoteneuronal stem cell differentiation, promote axonal and dendriticoutgrowth, and promote synaptic transmission, thereby relieving adversesymptoms of ASDs.

Compounds of the Invention

While the broadest definition of the invention is set out in theSummary, certain compounds of this invention are presently described.

In one aspect, this invention provides compounds of Formula 1 andFormula 2:

where m is 0 or 1;n is 0 or 1;X is H or —NR⁶R⁷;Y is H, alkyl, —CO₂R⁵, or —CONR⁶R⁷;Z is H, alkyl, —CO₂R⁵ or —CONR⁶R⁷;R¹ is H, alkyl, or aralkyl;R², R³, and R⁴ are independently H or alkyl;each R⁵ is independently H, alkyl, or a fatty alcohol residue;each R⁶ and R⁷ is independently H, alkyl, or aralkyl, or —NR⁶R⁷ ispyrrolidino, piperidino, or morpholino;or a lactone formed when a compound where Y is —CO₂(alkyl) and Z is—CO₂H or where Y is —CO₂H and Z is —CO₂(alkyl) is lactonized;and the pharmaceutically acceptable salts thereof,provided that the compound is not GPE, N-Me-GPE, GPE amide, APE, GPQ ora salt thereof.

In some aspects, this invention includes:

(a) the compounds are compounds of Formula 1;

(b) m is 0;

(c) n is 1;

(d) at least one of X, Y, R¹, R², R³, R⁴, and R⁵ is not hydrogen;

(e) X is —NR⁶R⁷; and

(f) Y is —CO₂R⁵ or —CO₂NR⁶R⁷ and

(g) Z is —CO₂R or —CO₂NR⁶R⁷.

Other compounds of the invention are compounds of Formula 1 wherein X is—NR⁶R⁷ and R⁶ and R⁷ are independently alkyl or aralkyl. The morepreferred embodiment is a compound of Formula 1 wherein X is —NR⁶R⁷ andboth R⁶ and R⁷ are alkyl.

Yet another compound of the invention is G-2-MePE, a compound of Formula1 wherein m is 0, n is 1, R1=R3=R4=H, R2 is methyl, X is NR⁶R⁷ whereR⁶=R⁷=H, Y is CO₂R⁵ where R⁵=H, Z is CO₂R⁵ where R⁵=H.

Pharmacology and Utility

Compounds of this invention can have anti-inflammatory, anti-apoptotic,anti-necrotic and neuroprotective effects. Their activity in vivo can bemeasured by cell counts, specific staining of desired markers, or bymethods such as those discussed in Klempt N D et al: Hypoxia-ischemiainduces transforming growth factor β1 mRNA in the infant rat brain.Molecular Brain, Research: 13: 93-101. Their activity can also bemeasured in vitro using methods known in the art or described herein.

Conditions affecting brain function become prevalent in agingpopulations. Memory loss and memory impairment are distressing topatients affected and their families. Memory loss or impairment canresult from normal aging, injury to the brain, neurodegenerative diseaseand psychological or psychiatric dysfunctions. It is therefore of greatbenefit to patients, their families and to society that novel compoundsare identified and characterized that enhance memory and/or cognitivefunction, and treat or prevent memory loss or impairment.

It is desirable to study effects of potential therapeutic agents inanimal systems. One such useful system is the rat. It is known that withaging, rats and other animals (including human beings) can exhibitsymptoms of memory loss, memory impairment and other cognitivedysfunctions. Further, it is known that studies in rats of therapeuticagents are predictive of therapeutic effects in humans. Thus, studies ofeffects of GPE and G-2-MePE and cognitive function in aging rats arereasonably predictive of therapeutic effects of those agents in aginghuman beings that have or are prone to acquiring memory deficits orother cognitive dysfunction. Compounds of this invention can enhancecognitive function and/or treat memory disorders. The cognitiveenhancing activity and therapeutic activity in vivo can be measured bystandard neuropsychological or behavioral tests known to individualsskilled in the art. Such tests can be chosen from a wide range ofavailable tests described above, and will vary depending on thecognitive function to be tested and the condition of the animal.

Standard behavioral tests useful for testing cognitive function inexperimental animals include but are not limited to the Morris WaterMaze test, passive avoidance response test, novel object recognitiontest, olfactory discrimination test, the 8-arm radial maze test and theT-maze test. These tests are directly applicable to studies of effectsof GPE and G-2-MePE on cognitive function in aging rats.

The compounds of this invention are also expected to havepharmacological and therapeutic activities similar to those of GPE, andthese activities may be measured by the methods known in the art, anddiscussed in the documents cited herein, and by methods used formeasuring the activity of GPE.

The therapeutic ratio of a compound can be determined, for example, bycomparing the dose that gives effective anti-inflammatory,anti-apoptotic and anti-necrotic activity in a suitable in vivo modelsuch as a hypoxic-ischemic injury (Sirimanne E S, Guan J, Williams C Eand Gluckman P D: Two models for determining the mechanisms of damageand repair after hypoxic-ischemic injury in the developing rat brain(Journal of Neuroscience Methods: 55: 7-14, 1994) in a suitable animalspecies such as the rat, with the dose that gives significant observableside-effects in the test animal species.

The therapeutic ratio of a compound can also be determined, for exampleby comparing the dose that gives effective cognitive functionenhancement or treats a memory disorder in a suitable in vivo model(Examples 4, 5 and 6 below) in a suitable animal species such as therat, with the dose that gives significant weight loss (or otherobservable side-effects) in the test animal species.

Compounds of this invention can be useful in treatment of a variety ofneurodegenerative disorders, including hypoxia/ischemia and neuronaldegeneration (U.S. Pat. No. 7,041,314), traumatic brain injury, motordisorders and seizures, stroke, and cardiac artery bypass graft surgery(U.S. Pat. No. 7,605,177), non-convulsive seizures (U.S. Pat. No.7,714,020), and disorders of cognitive function (U.S. application Ser.No. 12/903,844). Additionally, as described more fully herein below,compounds of this invention can be useful for treating Rett Syndrome,including prolonging life, increasing neuronal activity and treatingseizures associated with Rett Syndrome.

In one study of Rett Syndrome in mice (using the MeCP2 knock-out model),GPE was found to have effects to prolong life and increase neuronalfunction (U.S. Publication No. 2009/0099077). However, as disclosedfurther herein, GPE, being a naturally occurring peptide, is rapidlydegraded in vivo and in vitro, and its utility in chronic therapy ofpatients with Rett Syndrome is therefore unclear.

Pharmaceutical Compositions and Administration

In general, compounds of this invention can be administered intherapeutically effective amounts by any of the usual modes known in theart, either singly or in combination with at least one other compound ofthis invention and/or at least one other conventional therapeutic agentfor the disease being treated. A therapeutically effective amount mayvary widely depending on the disease or injury, the severity of thedisease, the age and relative health of the animal being treated, thepotency of the compound(s), and other factors. As anti-inflammatory,anti-apoptotic, anti-necrotic, anti-neurodegenerative, therapeuticallyeffective amounts of compounds of this invention can range from about0.001 milligrams per kilogram (mg/kg) to about 100 (mg/kg) mass of theanimal, for example, about 0.1 to about 10 mg/kg, with lower doses suchas about 0.001 to about 0.1 mg/Kg, e.g. about 0.01 mg/Kg, beingappropriate for administration through the cerebrospinal fluid, such asby intracerebroventricular administration, and higher doses such asabout 1 to about 100 mg/Kg, e.g. about 10 mg/Kg, being appropriate foradministration by methods such as oral, systemic (e.g. transdermal), orparenteral (e.g. intravenous) administration. A person of ordinary skillin the art will be able without undue experimentation, having regard tothat skill and this disclosure, to determine a therapeutically effectiveamount of a compound of this invention for a given disease or injury.

In general, compounds of this invention can be administered aspharmaceutical compositions by one of the following routes: oral,topical, systemic (e.g. transdermal, intranasal, or by suppository), orparenteral (e.g. intramuscular, subcutaneous, or intravenous injection),by administration to the CNS (e.g. by intraspinal or intercisternalinjection); by implantation, and by infusion through such devices asosmotic pumps, implantable pumps, transdermal patches, and the like.Compositions can take the form of tablets, pills, capsules, semisolids,powders, sustained release formulation, solutions, suspensions, elixirs,aerosols, soluble gels or any other appropriate compositions; andcomprise at least one compound of this invention in combination with atleast one pharmaceutically acceptable or physiological acceptableexcipient. Suitable excipients are well known to persons of ordinaryskill in the art, and they, and the methods of formulating thecompositions, may be found in such standard references as Gennaro AR:Remington: The Science and Practice of Pharmacy, 20^(th) ed.,Lippincott, Williams & Wilkins, 2000. Suitable liquid carriers,especially for injectable solutions, include water, aqueous salinesolution, aqueous dextrose solution, glycols, and the like, withisotonic solutions being preferred for intravenous, intraspinal, andintracisternal administration and vehicles such as artificialcerebrospinal fluid being also especially suitable for administration ofthe compound to the CNS. The above text is expressly incorporated hereinfully by reference.

Compounds of this invention can be administered orally, in tablets orcapsules. In some embodiments, compounds of this invention can beprepared in water-in-oil emulsions in the form of microemulsions, coarseemulsions, liquid crystals, or nanocapsules (U.S. application Ser. No.12/283,684, now U.S. Pat. No. 7,887,839 issued Feb. 15, 2011). Becausecompounds of this invention can have substantial oral bioavailability,they can be advantageously used for convenient and chronicadministration. Additionally, orally available compositions includesoluble hydrogels containing active compounds, thus permitting oraladministration of neuroprotective compounds without the need for apatient to swallow a tablet or capsule. Such slow-release materials andgels are known in the art.

Compounds of this invention can be administered after or before onset ofa condition that is likely to result in neurodegeneration or a symptomthereof. For example, it is known that hypoxia/ischemia can occur duringcoronary artery bypass graft (CABG) surgery. Thus, a patient can bepre-treated with a compound of this invention before being placed on anextracorporeal oxygenation system. In some embodiments, it can bedesirable to administer a compound of this invention beginning about 4hours before surgery or before an event that is likely to lead totraumatic or other neurological injury. In other embodiments, it can bedesirable to infuse a compound of this invention during the surgery orduring a surgical procedure to repair a neurological injury. Compoundsof this invention can also be used in emergency situations, for examplein a patient that has just experienced a stroke, hypoxic event,traumatic brain injury or other acute insult. In such situations, acompound of this invention can be administered immediately after adiagnosis of neural injury is made.

In some situations, kits containing compound of this invention can beprepared in advance of use in the field. A kit can contain a vialcontaining a compound of the invention in a pharmaceutically acceptableformulation (e.g., for injection or oral administration), along with asyringe or other delivery device, and instructions for use. Insituations in which a seizure is diagnosed, a compound of this inventioncan be administered along with an anticonvulsant, Many anticonvulsantsare known in the art and need not be described in detail herein.

Additionally, “secondary” neurological injuries can occur after aprimary insult such as a traumatic injury, stroke or surgical procedure.For example, after a stroke, penetrating brain injury or a CABGprocedure, inflammation of neural tissue can lead to neurodegeneration.Secondary injuries can be reflected by increased activation ofinflammatory cells (e.g., astrocytes and/or microglia), and actions ofinflammatory mediators can cause neurological damage. Thus, in someembodiments, it can be desirable to administer a compound of thisinvention for periods beginning before the insult, to up to about 100hours after the insult. In other embodiments, it can be desirable toadminister a compound of this invention beginning before the insult,during the insult and after the insult, either continuously, as aninfusion, or in discrete doses separated by a desired time interval.

Compounds of this invention can also be suitably administered by asustained-release system or gel material with G-2-MePE incorporatedtherein. Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., 1983), poly(2-hydroxyethylmethacrylate) (Langer et al., 1981), ethylene vinyl acetate (Langer etal., supra), or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Additionally, gel compositions based on polysaccharides (e.g.,carboxymethyl cellulose, carboxyethyl cellulose, chitosan or othercellulose derivatives) and polyethylene oxide derivatives (e.g.,polyethylene glycols) can be used used. These gel compositions aresoluble in aqueous solutions, are biocompatible, non-toxic and thereforecan be used for administering compounds of this invention to any mucosalsurface, including the oral cavity, nasopharynx, urogenital tract,intestine or rectum.

Sustained-release compositions also include a liposomally entrappedcompound. Liposomes containing the compound are prepared by methodsknown per se; DE 3,218,121; Epstein et al., 1985; Hwang et al., 1980; EP52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, liposomes are of the small (from or about 200 to 800Angstroms) unilamellar type in which the lipid content is greater thanabout 30 mole percent cholesterol, the selected proportion beingadjusted for the most efficacious therapy. Each and every of theabove-identified publications is expressly herein incorporated fully byreference, as if each had been separately so incorporated.

Compounds of this invention can also be attached to polyethylene glycol(“PEGylated”) to increase their lifetime in vivo, based on, e.g., theconjugate technology described in WO 95/32003.

Desirably, if possible, when administered as an anti-inflammatory, ananti-apoptotic agent, an anti-necrotic agent, or ananti-neurodegenerative agent, compounds of this invention can beadministered orally. The amount of a compound of this invention in thecomposition can vary widely depending on the type of composition, sizeof a unit dosage, kind of excipients, and other factors well known tothose of ordinary skill in the art. In general, the final compositioncan comprise from about 0.0001 percent by weight (% w) to about 10% w ofthe compound of this invention, preferably about 0.001% w to about 1% w,with the remainder being an excipient or excipients.

A composition may optionally contain, in addition to a compound of thisinvention, at least one agent selected from, for example, growth factorsand associated derivatives (insulin-like growth factor-1 (IGF-1),insulin-like growth factor-II (IGF-II), transforming growth factor-β1,activin, growth hormone, nerve growth factor, brain-derived neurotrophicfactor (BDNF), growth hormone binding protein, IGF-binding proteins(especially IGFBP-3), basic fibroblast growth factor, acidic fibroblastgrowth factor, the hst/Kfgk gene product, FGF-3, FGF-4, FGF-6,keratinocyte growth factor, androgen-induced growth factor. Additionalmembers of the FGF family include, for example, int-2, fibroblast growthfactor homologous factor-1 (FHF-1), FHP-2, FHF-3 and FHF-4, karatinocytegrowth factor 2, glial-activating factor, FGF-10 and FGF-16, ciliaryneurotrophic factor, brain derived growth factor, neurotrophin 3,neurotrophin 4, bone morphogenetic protein 2 (BMP-2), glial-cell linederived neurotrophic factor, activity-dependant neurotrophic factor,cytokine leukaemia inhibiting factor, oncostatin M, interleukin), α-,β-, γ-, or consensus interferon, and TNF-α. Other forms ofneuroprotective therapeutic agents include, for example, clomethiazole;kynurenic acid, Semax, tacrolimus,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol,andrenocorticotropin-(4-9) analog [ORG 2766] and dizolcipine (MK-801),selegiline; glutamate antagonists such as mematine (Namenda) NPS1506,GV1505260, MK-801, GV150526; AMPA antagonists such as2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070and LY300164; anti-inflammatory agents directed against the addressinMAdCAM-1 and/or its integrin α4 receptors (α4β1 and α4β7), such asanti-MAdCAM-1mAb MECA-367 (ATCC accession no. HB-9478). Combinationtherapy with metabotropic glutamate receptor antagonists such as fenobammay also be useful. Also, in addition to a compound of this invention, acomposition may include a selective serotonin reuptake inhibitor such asfluoxetine, a selective norepinephine reuptake inhibitor such asviloxazine, or an atypical anti-psychotic such as risperidone. Most ofthese agents, especially the peptides such as the growth factors, etc.,are not orally active, and will require administration by injection orinfusion.

Preparation of Compositions

The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCompany (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis,Mo.), or are prepared by methods well known to the person of ordinaryskill in the art following procedures described in such references asFieser and Fieser's Reagents for Organic Synthesis, vols 1-17, JohnWiley and Sons, New York, N.Y., 1991; Rodd's Chemistry of CarbonCompounds, vols. 1-5 and supplements, Elsevier Science Publishers, 1989;Organic Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y.,1991; March J; Advanced Organic Chemistry, 4^(th) ed. John Wiley andSons, New York, N.Y., 1992; and Larock: Comprehensive OrganicTransformations, VCH Publishers, 1989. In most instances, amino acidsand their esters or amides, and protected amino acids, are widelycommercially available; and the preparation of modified amino acids andtheir amides or esters are extensively described in the chemical andbiochemical literature and thus well-known to persons of ordinary skillin the art. For example, N-pyrrolidineacetic acid is described inDega-Szafran Z and Pryzbylak R. Synthesis, IR, and NMR studies ofzwitterionic α-(1-pyrrolidine)alkanocarboxylic acids and their N-methylderivatives. J. Mol. Struct.: 436-7, 107-121, 1997; andN-piperidineacetic acid is described in Matsuda O, Ito S, and Sekiya M.Reaction of N-(alkoxymethyl)dialkylamines andN,N′-methylenebisdialkylamines with isocyanides. Chem. Pharm. Bull.:23(1), 219-221, 1975. Each of the above-identified publications isherein expressly incorporated fully by reference as though individuallyso incorporated.

Starting materials, intermediates, and compounds of this invention maybe isolated and purified using conventional techniques, includingfiltration, distillation, crystallization, chromatography, and the like.They may be characterized using conventional methods, including physicalconstants and spectral data.

Compounds of this invention may be prepared by the methods describedbelow and as given in the Examples.

Compounds of Formula 1 are analogs of GPE, or modifications thereof,such as esters or amides. In general, they may be prepared by methodssuch as are already well-known to persons of ordinary skill in the artof peptide and modified peptide synthesis, following the reactionschemes set forth in the FIGS. 1-11 accompanying this specification, orby following other methods well-known to those of ordinary skill in theart of the synthesis of peptides and analogs.

Conveniently, synthetic production of the polypeptides of the inventionmay be according to the solid-phase synthetic method described byMerrifield et al. Solid phase peptide synthesis. I. The synthesis of atetrapeptide: J. Amer. Chem. Soc.; 85, 2149-2156, 1963. This techniqueis well understood and is a common method for preparation of peptides.The general concept of this method depends on attachment of the firstamino acid of the chain to a solid polymer by a covalent bond.Succeeding protected amino acids are added, one at a time (stepwisestrategy), or in blocks (segment strategy), until the desired sequenceis assembled. Finally, the protected peptide is removed from the solidresin support and the protecting groups are cleaved off. By thisprocedure, reagents and by-products are removed by filtration, thuseliminating the necessity of purifying intermediaries.

Amino acids may be attached to any suitable polymer as a resin. Theresin must contain a functional group to which the first protected aminoacid can be firmly linked by a covalent bond. Various polymers aresuitable for this purpose, such as cellulose, polyvinyl alcohol,polymethylmethacrylate and polystyrene. Suitable resins are commerciallyavailable and well known to those of skill in the art. Appropriateprotective groups usable in such synthesis include tert-butyloxycarbonyl(BOC), benzyl (Bzl), t-amyloxycarbonyl (Aoc), tosyl (Tos),o-bromo-phenylmethoxycarbonyl (BrZ), 2,6-dichlorobenzyl (BzlCl₂), andphenylmethoxycarbonyl (Z or CBZ). Additional protective groups areidentified in Merrifield, cited above, as well as in McOmie J F W:Protective Groups in Organic Chemistry, Plenum Press, New York, 1973,both references expressly incorporated fully herein.

General procedures for preparing peptides of this invention involveinitially attaching a carboxyl-terminal protected amino acid to theresin. After attachment the resin is filtered, washed and the protectinggroup (desirably BOC) on the 1-amino group of the carboxyl-terminalamino acid is removed. The removal of this protecting group must takeplace, of course, without breaking the bond between that amino acid andthe resin. The next amino, and if necessary, side chain protected aminoacid, is then coupled to the free I-amino group of the amino acid on theresin. This coupling takes place by the formation of an amide bondbetween the free carboxyl group of the second amino acid and the aminogroup of the first amino acid attached to the resin. This sequence ofevents is repeated with successive amino acids until all amino acids areattached to the resin. Finally, the protected peptide is cleaved fromthe resin and the protecting groups removed to reveal the desiredpeptide. The cleavage techniques used to separate the peptide from theresin and to remove the protecting groups depend upon the selection ofresin and protecting groups and are known to those familiar with the artof peptide synthesis.

Alternative techniques for peptide synthesis are described in Bodanszkyet al, Peptide Synthesis, 2nd ed, John Wiley and Sons, New York, 1976.For example, the peptides of the invention may also be synthesized usingstandard solution peptide synthesis methodologies, involving eitherstepwise or block coupling of amino acids or peptide fragments usingchemical or enzymatic methods of amide bond formation. (See, e.g., H. D.Jakubke in The Peptides, Analysis, Synthesis, Biology, Academic Press,New York, 1987, p. 103-165; J. D. Glass, ibid., pp. 167-184; andEuropean Patent 0324659 A2, describing enzymatic peptide synthesismethods.) These solution synthesis methods are well known in the art.Each of the above-identified publications is expressly incorporatedherein fully by reference as though individually so incorporated.

Commercial peptide synthesizers, such as the Applied Biosystems Model430A, are available for the practice of these methods.

A person of ordinary skill in the art will not have to undertake undueexperimentation, taking account of that skill and the knowledgeavailable, and of this disclosure, in developing one or more suitablesynthetic methods for compounds of this invention.

For example, analogs in which the glycine residue of GPE is replaced byan alternative amino acid, or by a non-amino acid, may conveniently beprepared by the preparation of a C-protected proline-glutamic aciddipeptide (such as the dibenzyl ester), and coupling that dipeptide withan N-protected glycine analog, such as BOC-N-methylglycine,BOC-L-valine, N-pyrrolidineacetic acid, and the like, followed bydeprotection, as illustrated in FIGS. 2 and 3. Analogs in which theglutamic acid residue of GPE is replaced by an alternative amino acid oran amino acid amide or ester may conveniently be prepared by thepreparation of an N-protected glycine-L-proline dipeptide (such asBOC-glycyl-L-proline), and coupling that dipeptide with a C-protectedglutamic acid or analog thereof, such as tert-butyl γ-aminobutyrate,methyl 4-amino-4-dimethylcarbamoylbutyrate, L-glutamine methyl ester,dimethyl I-methylglutamate, etc. Lactones may be prepared by thepreparation of an appropriate mono-acid-mono-ester derivative andreduction Analogs in which R² is alkyl may conveniently be preparedsimply by use of the appropriate 2-alkylproline in the synthesis, andsimilarly analogs in which R³ is alkyl may conveniently be prepared bythe use of the appropriate N-alkylglutamic acid or analog in thesynthesis. Where modifications are to be made to two or more aminoacids, the coupling techniques will still be the same, with just morethan one modified amino acid or analog being used in the synthesis. Thechoice of appropriate protecting groups for the method chosen(solid-phase or solution-phase), and of appropriate substrates ifsolid-phase synthesis is used, will be within the skill of a person ofordinary skill in the art.

Compounds of Formula 2 may be prepared from suitably protected5-oxo-L-proline or analogs or derivatives thereof, following methodssuch as the coupling of the proline carboxyl group with a protectedglutamic acid or analog or derivative to give an analog of intermediateA of FIG. 2, comparable to the coupling reaction shown in FIG. 2, andthen alkylating the pyrrolidine nitrogen with a group of the formulaA-(CH₂)_(m)—CH(R¹)—CH₂R, protected at A if necessary, where R is aleaving group under alkylation conditions. Alternatively, the suitablyprotected 5-oxo-L-proline may first by alkylated at the pyrrolidinenitrogen to give an analog of intermediate B of FIG. 4, and thencoupling this with a suitably protected glutamic acid or analog orderivative in the manner shown in FIGS. 4 though 9.

EXAMPLES

The following examples are intended to illustrate embodiments of thisinvention, and are not intended to limit the scope to these specificexamples. Persons of ordinary skill in the art can apply the disclosuresand teachings presented herein to develop other embodiments withoutundue experimentation and with a likelihood of success. All suchembodiments are considered part of this invention.

Example 1 Synthesis of N,N-Dimethylglycyl-L-prolyl)-L-glutamic acid

The following non-limiting example illustrates the synthesis of acompound of the invention, N,N-Dimethylglycyl-L-prolyl-L-glutamic acid

All starting materials and other reagents were purchased from Aldrich;BOC=tert-butoxycarbonyl; Bn=benzyl.

BOC-L-proline-(β-benzyl)-L-glutamic acid benzyl ester

To a solution of BOC-proline [Anderson G W and McGregor A C: J. Amer.Chem. Soc.: 79, 6810, 1994] (10 mmol) in dichloromethane (50 ml), cooledto 0° C., was added triethylamine (1.39 ml, 10 mmol) and ethylchloroformate (0.96 ml, 10 mmol). The resultant mixture was stirred at0° C. for 30 minutes. A solution of dibenzyl-L-glutamate (10 mmol) wasthen added and the mixture stirred at 0° C. for 2 hours then warmed toroom temperature and stirred overnight. The reaction mixture was washedwith aqueous sodium bicarbonate and citric acid (2 mol l⁻¹) then dried(MgSO₄) and concentrated at reduced pressure to giveBOC-L-proline-L-glutamic acid dibenzyl ester (5.0 g, 95%).

L-proline-L-glutamic acid dibenzyl ester

A solution of BOC-L-glutamyl-L-proline dibenzyl ester (3.4 g, 10 mmol),cooled to 0° C., was treated with trifluoroacetic acid (25 ml) for 2 h.at room temperature. After removal of the volatiles at reduced pressurethe residue was triturated with ether to give L-proline-L-glutamic aciddibenzyl ester.

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid

A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane(10 ml) was added to a stirred and cooled (0° C.) solution ofL-proline-L-glutamic acid dibenzyl ester (10 mmol), N,N-dimethylglycine(10 mmol) and triethylamine (10.3 mmol) in dichloromethane (30 ml). Themixture was stirred at 0° C. overnight and then at room temperature for3 h. After filtration, the filtrate was evaporated at reduced pressure.The resulting crude dibenzyl ester was dissolved in a mixture of ethylacetate (30 ml) and methanol (30 ml) containing 10% palladium oncharcoal (0.5 g) then hydrogenated at room temperature and pressureuntil the uptake of hydrogen ceased. The filtered solution wasevaporated and the residue recrystallised from ethyl acetate to yieldthe tripeptide derivative.

It can be appreciated that following the method of the Examples, andusing alternative amino acids or their amides or esters, will yieldother compounds of Formula 1.

Example 2 Synthesis of Glycyl-L-2-Methyl-L-Prolyl-L-GlutamateGlycl-L-2-Methylprolyl-L-Glutamic Acid (G-2MePE)

L-2-Methylproline and L-glutamic acid dibenzyl ester p-toluenesulphonatewere purchased from Bachem, N-benzyloxycarbonyl-glycine from AcrosOrganics and bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl, 97%)from Aldrich Chem. Co.

Methyl L-2-methylprolinate hydrochloride 2

Thionyl chloride (5.84 cm³, 80.1 mmol) was cautiously added dropwise toa stirred solution of (L)-2-methylproline 1 (0.43 g, 3.33 mmol) inanhydrous methanol (30 cm³) at −5° C. under an atmosphere of nitrogen.The reaction mixture was heated under reflux for 24 h, and the resultantpale yellow-coloured solution was concentrated to dryness in vacuo. Theresidue was dissolved in a 1:1 mixture of methanol and toluene (30 cm³)then concentrated to dryness to remove residual thionyl chloride. Thisprocedure was repeated twice more, yielding hydrochloride 2 (0.62 g,104%) as an hygroscopic, spectroscopically pure, off-white solid: mp127-131° C.; [α]_(D) −59.8 (c 0.24 in CH₂Cl₂); ν_(max) (film)/cm⁻¹ 3579,3398 br, 2885, 2717, 2681, 2623, 2507, 1743, 1584, 1447, 1432, 1374,1317, 1294, 1237, 1212, 1172, 1123, 981, 894, 861 and 764; δ_(H) (300MHz; CDCl₃; Me₄Si) 1.88 (3H, s, Proα-CH₃), 1.70-2.30 (3H, br m,Proβ-H_(A)H_(B) and Proγ-H₂), 2.30-2.60 (1H, br m, Proβ-H_(A)H_(B)),3.40-3.84 (2H, br m, Proδ-H₂), 3.87 (3H, s, CO₂CH₃), 9.43 (1H, br s, NH)and 10.49 (1H, br s, HCl); δ_(C) (75 MHz; CDCl₃) 21.1 (CH₃, Proα-CH₃),22.4 (CH₂, Proγ-C), 35.6 (CH₂, Proβ-C), 45.2 (CH₂, Proδ-C), 53.7 (CH₃,CO₂CH₃), 68.4 (quat., Proα-C) and 170.7 (quat., CO); m/z (FAB+) 323.1745[M₂.H³⁵Cl.H⁺: (C₇H₁₃NO₂)₂.H³⁵Cl.H requires 323.1738] and 325.1718[M₂.H³⁷Cl.H⁺: (C₇H₁₃NO₂)₂.H³⁷Cl.H requires 325.1708].

N-Benzyloxycarbonyl-glycyl-L-2-methylproline 5

Anhydrous triethylamine (0.45 cm³, 3.23 mmol) was added dropwise to amixture of methyl L-2-methylprolinate hydrochloride 2 (0.42 g, 2.34mmol) and N-benzyloxycarbonyl-glycine (98.5%) 3 (0.52 g, 2.45 mmol) inmethylene chloride (16 cm³), at 0° C., under an atmosphere of nitrogen.The resultant solution was stirred for 20 min and a solution of1,3-dicyclohexylcarbodiimide (0.56 g, 2.71 mmol) in methylene chloride(8 cm³) at 0° C. was added dropwise and the reaction mixture was warmedto room temperature and stirred for a further 20 h. The resultant whitemixture was filtered through a Celite™ pad to partially remove1,3-dicyclohexylurea, and the pad was washed with methylene chloride (50cm³). The filtrate was washed successively with 10% aqueous hydrochloricacid (50 cm³) and saturated aqueous sodium hydrogen carbonate (50 cm³),dried (MgSO₄), filtered, and concentrated to dryness in vacuo. Furtherpurification of the residue by flash column chromatography (35 g SiO₂;30-70% ethyl acetate-hexane; gradient elution) afforded tentativelymethyl N-benzyloxycarbonyl-glycyl-L-2-methylprolinate 4 (0.56 g),containing 1,3-dicyclohexylurea, as a white semi-solid: R_(f) 0.65(EtOAc); m/z (EI+) 334.1534 (M⁺.C₁₇H₂₂N₂O₅ requires 334.1529) and 224(1,3-dicyclohexylurea).

To a solution of impure prolinate 4 (0.56 g, ca. 1.67 mmol) in1,4-dioxane (33 cm³) was added dropwise 1M aqueous sodium hydroxide (10cm³, 10 mmol) and the mixture was stirred for 19 h at room temperature.Methylene chloride (100 cm³) was then added and the organic layerextracted with saturated aqueous sodium hydrogen carbonate (2×100 cm³).The combined aqueous layers were carefully acidified with hydrochloricacid (32%), extracted with methylene chloride (2×100 cm³), and thecombined organic layers dried (MgSO₄), filtered, and concentrated todryness in vacuo. Purification of the ensuing residue (0.47 g) by flashcolumn chromatography (17 g SiO₂; 50% ethyl acetate-hexane to 30%methanol-dichloromethane; gradient elution) gave N-protected dipeptide 5(0.45 g, 60%) as a white foam in two steps from hydrochloride 2.Dipeptide 5 was shown to be exclusively the trans-orientated conformerby NMR analysis: R_(f) 0.50 (20% MeOH—CH₂Cl₂); [α]_(D) −62.3 (c 0.20 inCH₂Cl₂); ν_(max) (film)/cm⁻¹ 3583, 3324 br, 2980, 2942, 1722, 1649,1529, 1454, 1432, 1373, 1337, 1251, 1219, 1179, 1053, 1027, 965, 912,735 and 698; δ_(H) (300 MHz; CDCl₃; Me₄Si) 1.59 (3H, s, Proα-CH₃), 1.89(1H, 6 lines, J 18.8, 6.2 and 6.2, Prop-H_(A)H_(B)), 2.01 (2H, dtt, J18.7, 6.2 and 6.2, Proγ-H₂), 2.25-2.40 (1H, m, Proβ-H_(A)H_(B)), 3.54(2H, t, J 6.6, Proδ-H₂), 3.89 (1H, dd; J 17.1 and 3.9, Glyα-H_(A)H_(B)),4.04 (1H, dd, J 17.2 and 5.3, Glyα-H_(A)H_(B)), 5.11 (2H, s, OCH₂Ph),5.84 (1H, br t, J 4.2, N—H), 7.22-7.43 (5H, m, Ph) and 7.89 (1H, br s,—COOH); δ_(C) (75 MHz; CDCl₃) 21.3 (CH₃, Proα-CH₃), 23.8 (CH₂, Proγ-C),38.2 (CH₂, Prop-C), 43.6 (CH₂, Glyα-C), 47.2 (CH₂, Proδ-C), 66.7 (quat,Proα-C), 66.8 (CH₂, OCH₂Ph), 127.9 (CH, Ph), 127.9 (CH, Ph), 128.4, (CH,Ph), 136.4 (quat., Ph), 156.4 (quat., NCO₂), 167.5 (quat., Gly-CON) and176.7 (quat., CO); m/z (EI+) 320.1368 (M⁺. C₁₆H₂₀N₂O₅ requires320.1372).

Dibenzyl N-benzyloxycarbonyl-glycyl-L-2-methylprolyl-L-glutamate 7

Triethylamine (0.50 cm³, 3.59 mmol) was added dropwise to a solution ofdipeptide 5 (0.36 g, 1.12 mmol) and L-glutamic acid dibenzyl esterp-toluenesulphonate 6 (0.73 g, 1.46 mmol) in methylene chloride (60 cm³)under nitrogen at room temperature, and the reaction mixture stirred for10 min. Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl, 97%) (0.37g, 1.41 mmol) was added and the colourless solution stirred for 17 h.The methylene chloride solution was washed successively with 10% aqueoushydrochloric acid (50 cm³) and saturated aqueous sodium hydrogencarbonate (50 cm³), dried (MgSO₄), filtered, and evaporated to drynessin vacuo. Purification of the resultant residue by repeated (2×) flashcolumn chromatography (24 g SiO₂; 30-70% ethyl acetate-hexane; gradientelution) yielded fully protected tripeptide 7 (0.63 g, 89%) as acolourless oil, Tripeptide 7 was shown to be exclusively thetrans-orientated conformer by NMR analysis: R_(f) 0.55 (EtOAc); [α]_(D)−41.9 (c 0.29 in CH₂Cl₂); ν_(max) (film)/cm⁻¹ 3583, 3353 br, 2950, 1734,1660, 1521, 1499, 1454, 1429, 1257, 1214, 1188, 1166, 1051, 911, 737 and697; δ_(H) (400 MHz; CDCl₃; Me₄Si) 1.64 (3H, s, Proα-CH₃), 1.72 (1H, dt,J 12.8, 7.6 and 7.6, Prop-H_(A)H_(B)), 1.92 (2H, 5 lines, J 6.7,Proγ-H₂), 2.04 (1H, 6 lines, J 7.3 Gluβ-H_(A)H_(B)), 2.17-2.27 (1H, m,Gluβ-H_(A)H_(B)), 2.35-2.51 (3H, m, Prop-H_(A)H_(B) and Gluγ-H₂),3.37-3.57 (2H, m, Proδ-H₂), 3.90 (1H, dd, J 17.0 and 3.6,Glyα-H_(A)H_(B)), 4.00 (1H, dd, J 17.1 and 5.1, Glyα-H_(A)H_(B)), 4.56(1H, td, J 7.7 and 4.9, Gluα-H), 5.05-5.20 (6H, m, 3×OCH₂Ph), 5.66-5.72(1H, br m, Gly-NH), 7.26-7.37 (15H, m, 3×Ph) and 7.44 (1H, d, J 7.2,Glu-NH); δ_(C) (100 MHz; CDCl₃) 21.9 (CH₃, Proα-CH₃), 23.4 (CH₂,Proγ-C), 26.6 (CH₂, Gluβ-C), 30.1 (CH₂, Gluγ-C), 38.3 (CH₂, Prop-C),43.9 (CH₂, Glyα-C), 47.6 (CH₂, Proδ-C), 52.2 (CH, Gluα-C), 66.4 (CH₂,OCH₂Ph), 66.8 (CH₂, OCH₂Ph), 67.1 (CH₂, OCH₂Ph), 68.2 (quat, Proα-C),127.9 (CH, Ph), 128.0 (CH, Ph), 128.1, (CH, Ph), 128.2, (CH, Ph), 128.2,(CH, Ph), 128.3, (CH, Ph), 128.4, (CH, Ph), 128.5, (CH, Ph), 128.5, (CH,Ph), 135.2 (quat., Ph), 135.7 (quat., Ph), 136.4 (quat., Ph), 156.1(quat., NCO₂), 167.3 (quat., Gly-CO), 171.4 (quat., CO), 172.9 (quat.,CO) and 173.4 (quat., CO); m/z (FAB+) 630.2809 (MH⁺. C₃₅H₄₀N₃O₈ requires630.2815).

Glycyl-L-2-methylprolyl-L-glutamic acid (G-2-MePE)

A mixture of the protected tripeptide 7 (0.63 g, 1.00 mmol) and 10 wt. %palladium on activated carbon (0.32 g, 0.30 mmol) in 91:9 methanol-water(22 cm³) was stirred under an atmosphere of hydrogen at roomtemperature, protected from light, for 23 h. The reaction mixture wasfiltered through a Celite™ pad and the pad washed with 75:25methanol-water (200 cm³). The filtrate was concentrated to dryness underreduced pressure and the residue triturated with anhydrous diethyl etherto afford a 38:1 mixture of G-2-MePE and tentatively methylamine 8 (0.27g, 86%) as an extremely hygroscopic white solid. Analyticalreverse-phase HPLC studies on the mixture [Altech Econosphere C₁₈ Sicolumn, 150×4.6 mm, 5 □m; 5 min flush with H₂O (0.05% TFA) then steadygradient over 25 min to MeCN as eluent at flow rate of 1 ml/min;detection using diode array] indicated it was a 38:1 mixture of twoeluting peaks with retention times of 13.64 and 14.44 min at 207 and 197nm, respectively. G-2-MePE was shown to be a 73:27 trans:cis mixture ofconformers by ¹H NMR analysis (the ratio was estimated from the relativeintensities of the double doublet and triplet at δ 4.18 and 3.71,assigned to the Gluα-H protons of the major and minor conformers,respectively): mp 144° C.^(φ); [α]_(D) −52.4 (c 0.19 in H₂O); δH (300MHz; D₂O; internal MeOH) 1.52 (3H, s, Proα-CH₃), 1.81-2.21 (6H, m,Prop-H₂, Proγ-H₂ and Gluβ-H₂), 2.34 (1.46H, t, J 7.2, Gluγ-H₂), 2.42*(0.54H, t, J 7.3, Gluγ-H₂), 3.50-3.66 (2H, m, Proδ-H₂), 3.71* (0.27H, t,J 6.2, Gluα-H), 3.85 (1H, d, J 16.6, Glyα-H_(A)H_(B)), 3.92 (1H, d, J16.6, Glyα-H_(A)H_(B)) and 4.18 (0.73H, dd, J 8.4 and 4.7, Gluα-H); δC(75 MHz; D₂O; internal MeOH) 21.8 (CH₃, Proα-CH₃), 25.0 (CH₂, Proγ-C),27.8* (CH₂, Gluβ-C), 28.8 (CH₂, Gluβ-C), 32.9 (CH₂, Gluγ-C), 40.8 (CH₂,Prop-C), 42.7 (CH₂, Glyα-C), 49.5 (CH₂, Proδ-C), 56.0* (CH, Gluα-C),56.4 (CH, Gluα-C), 69.8 (quat, Proα-C), 166.5 (quat., Gly-CO), 177.3(quat., Pro-CON), 179.2 (quat., Gluα-CO), 180.2* (quat., Gluγ-CO) and180.6 (quat., Gluγ-CO); m/z (FAB+) 316.1508 (MH⁺. C₁₃H₂₂N₃O₆ requires316.1509).

Example 3 In Vitro Neuroprotection

Therapeutic effects of GPE analogs were examined in a series ofexperiments in vitro to determine their effects on neurodegeneration ofneural cells of different origin. The in vitro systems described hereinare well-established in the art and are known to be predictive ofneuroprotective effects observed in vivo, including effects in humanssuffering from neurodegenerative disorders.

Material and Methods

The following experimental protocol followed guidelines approved by theUniversity of Auckland Animal Ethics Committee.

Preparation of Cortical Astrocyte Cultures for Harvest of MetabolisedCell Culture Supernatant

One cortical hemisphere from a postnatal day 1 rat was used andcollected into 4 ml of DMEM. Trituration was performed using a 5 mlglass pipette and an 18-gauge needle. The cell suspension was sievedthrough a 100 μm cell strainer and washed in 50 ml DMEM (centrifugationfor 5 min at 250 g). The sediment was resuspended in 20 ml DMEM+10%fetal calf serum. The suspension was added into two 25 cm³ flasks (10 mlper flask) and cultivated at 37° C. in the presence of 10% CO₂ followedby a change of the medium twice a week. When cells reached confluence,they were washed three times with PBS, adjusted to Neurobasal/B27 andincubated for another 3 days. This supernatant was frozen for transientstorage at −80° C.

Preparation of Stratial and Cortical Tissue from Rat E18/E19 Embryos

A dam was sacrificed by CO₂-treatment, and then was prepared forcaesarean section. After surgery, the embryos were removed from theiramniotic sacs and decapitated. The heads were placed on ice in DMEM/F12medium for striatum and PBS+0.65% D(+)-glucose for cortex.

Striatal Tissue Extraction Procedure and Preparation of Cells

A whole brain was removed from the skull with the ventral side facingupwards in DMEM/F12 medium. The striatum was dissected out from bothhemispheres under a stereomicroscope and the striatal tissue was placedinto a Falcon tube on ice. Striatal tissue was then triturated using aP1000 pipettor in 1 ml of volume. The tissue was triturated by gentlypipetting the solution up and down into the pipette tip about 15 times,using shearing force on alternate outflows. The tissue pieces settled tothe bottom of the Falcon tube within 30 seconds. The supernatantcontaining a suspension of dissociated single cells was then transferredto a new sterile Falcon tube on ice. The tissue pieces were trituratedagain to avoid excessively damaging already dissociated cells, by overtriturating them. 1 milliliter of ice-cold DMEM/F12 medium was added tothe tissue pieces in the first tube and triturated as before. The tissuepieces were allowed to settle and the supernatant was removed to a newsterile Falcon tube on ice. The cells were centrifuged at 250 g for 5minutes at 4° C.

Plating and Cultivation of Striatal Cells

Striatal cells were plated into Poly-L-Lysine (0.1 mg/ml) coated 96-wellplates (the inner 60 wells only) at a density of 200,000 cells/cm² inNeurobasal/B27 medium (Invitrogen). The cells were cultivated in thepresence of 5% CO₂ at 37° C. under 100% humidity. Medium was changed ondays 1, 3 and 6.

Cortical Tissue Extraction Procedure and Preparation of Cells

The two cortical hemispheres were carefully removed by spatula from thewhole brain with the ventral side facing upside into a PBS+0.65%D(+)-glucose containing petri dish. Forceps were put into the rostralpart (near B. olfactorius) of the cortex in order to fix the tissue andtwo lateral-sagittal oriented cuts were made to remove the paraform andentorhinal cortices. A frontal oriented cut at the posterior end wasmade to remove the hippocampal formation. A final frontal cut was done afew millimeters away from the last cut in order to get hold of area17/18 of the visual cortex.

Cortices were placed on ice in PBS+0.65%(+)-glucose and centrifuged at350 g for 5 minutes. The supernatant was removed and trypsin/EDTA(0.05%/0.53 mM) was added for 8 min at 37° C. The reaction was stoppedby adding an equal amount of DMEM and 10% fetal calf serum. Thesupernatant was removed by centrifugation followed by two subsequentwashes in Neurobasal/B27 medium.

The cells were triturated once with a glass Pasteur pipette in 1 ml ofNeurobasal/B27 medium and subsequently twice by using a 1 ml insulinsyringe with a 22 gauge needle. The cell suspension was passed through a100 μm cell strainer and rinsed by 1 ml of Neurobasal/B27 medium. Cellswere counted and adjusted to 50,000 cells per 60 μl.

Plating and Cultivation of Cortical Cells

96-well plates were coated with 0.2 mg/ml Poly-L-Lysine and subsequentlycoated with 2 μg/ml laminin in PBS, after which 60 μl of corticalastrocyte-conditioned medium was added to each well. Subsequently, 60 μlof cortical cell suspension was added. The cells were cultivated in thepresence of 10% CO₂ at 37° C. under 100% humidity. At day 1, there was acomplete medium change (1:1—Neurobasal/B27 and astrocyte-conditionedmedium) with addition of 1 μM cytosine-β-D-arabino-furanoside (mitosisinhibitor). On days 2 and 5, ⅔ of the medium was changed.

Cerebellar Microexplants from P8 Animals: Preparation, Cultivation andFixation

Laminated cerebellar cortices of the two hemispheres were explanted froma P8 rat, cut into small pieces in PBS+0.65% D(+) glucose solution andtriturated with a 23 gauge needle and subsequently pressed through a 125μm pore size sieve. The obtained microexplants were centrifuged (60 g)twice (media change) into serum-free BSA-supplemented STARTV-medium(Biochrom). For cultivation, 40 μl of cell suspension was adhered for 3hours on a 0.1 mg/ml Poly-L-Lysine coated cover slip placed in 35 mmsized 6 well plates in the presence of 5% CO₂ under 100% humidity at 34°C. Subsequently, 1 ml of STARTV-medium was added together with thetoxins and drugs. The cultures were monitored (evaluated) after 2-3 daysof cultivation in the presence of 5% CO₂ under 100% humidity. For cellcounting analysis, the cultures were fixed in rising concentrations ofparaformaldehyde (0.4%, 1.2%, 3% and 4% for 3 min each) followed by awash in PBS.

Toxin and Drug Administration to Neural Dells In Vitro and Analysis ofData

To study neuroprotective effects of GPE analogs, we carried out a seriesof experiments in vitro using okadaic acid to cause toxic injury toneural cells, Okadaic acid is an art-recognized toxin that is known tocause injury to neurons. Further, recovery of neural cells or neuralcell function after injury by okadaic acid is recognized to bepredictive of recoveries from injuries caused by other toxins.

To cause toxic injury to neurons, we exposed neurons to 1:100 parts ofokadaic acid at concentrations of 30 nM or 100 nM and 0.5 mM3-nitropropionic acid (for cerebellar microexplants only). GPE (1 nM-1mM) or G-2-MePE (1 nM-1 mM) was used at 8 days in vitro (DIV) forcortical cultures and 9DIV for striatal cultures. The incubation timewas 24 hours. The survival rate was determined by a colorimetricend-point MTT-assay at 595 nm in a multi-well plate reader. For thecerebellar microexplants four windows (field of 0.65 mm²) with highestcell density were chosen and cells displaying neurite outgrowth werecounted.

Results

The GPE analog G-2-MePE exhibited comparable neuroprotective effectswithin all three tested in vitro systems (FIGS. 12-15).

Cortical cultures responded to 10 μM concentrations of GPE (FIG. 12) orG-2-MePE (10 μM, FIG. 13) with 64% and 59% neuroprotection,respectively.

The other 2 types of cultures demonstrated neuroprotection at lowerdoses of G-2-MePE (cerebellar microexplants: FIG. 14 and striatal cells:FIG. 15). Striatal cells demonstrated neuroprotection within the rangeof 1 nM to 1 mM of G-2-MePE (FIG. 15), while the postnatal cerebellarmicroexplants demonstrated neuroprotection with G-2-MePE in the doserange between about 1 nM and about 100 nM (FIG. 14). Thus, we concludethat G-2-MePE is a neuroprotective agent and can have therapeuticeffects in humans suffering from neurodegenerative disorders. BecauseG-2-MePE can be neuroprotective when directly administered to neurons inculture, that G-2-MePE can be effective in vivo when directlyadministered to the brains of affected animals.

Example 4 Effects of G-2-MePE on Striatal Cholinergic Neurons in AgingRats

To determine whether G-2-MePE can affect cholinergic neurons, we studiedaging rats. Choline acetyltransferase (ChAT) is an enzyme that isinvolved in the biosynthesis of the neurotransmitter for cholinergicnerves, acetylcholine. It is well known that immunodetection of ChAT canbe used to determine the numbers of cholinergic nerves present in atissue. It is also known that the numbers of cholinergic nerves presentis associated with the physiological function of cholinergic neuralpathways in the brain.

In this experiment, we tested the effects of G-2-MePE on the number ofChAT-positive neurons in brains of 18-month old rats.

Methods

Eighteen-month old male rats received one of five treatments. A controlgroup was treated with vehicle (saline alone (n=4) and four groups weretreated with a single dose of G-2-MePE. Doses of 0.012 (n=4), 0.12(n=5), 1.2 (ln=5) and 12 mg/kg (n=3), respectively, were givensub-cutaneously. Rats were sacrificed with an overdose of pentobarbital3 days after drug treatment. Brains were perfused with normal saline and4% paraformaldehyde and fixed in perfusion fixative overnight. Brainswere stored in 25% sucrose in 0.1M PBS (pH7.4) until the tissue sank.Frozen coronal sections of striatum were cut with a microtome and storedin 0.1% sodium azide in 0.1M PBS at 4° C. Immunoreactivity for cholineacetyltransferase (ChAT) was established by staining using a freefloating section method. Briefly, antibodies were diluted in 1% goatserum. The sections were incubated in 0.2% triton in 0.1M PBS/Triton™ at4° C. overnight before Immunohistochemical staining. The sections werepre-treated with 1% H₂O₂ in 50% methanol for 20 min. The sections werethen incubated with rabbit (Rb) anti-ChAT (1:5000) antibodies (theprimary antibodies) in 4D on a shaker for two days. The sections werewashed using PBS/Triton™ (15 minutes×3d) and then incubated with goatanti-rabbit biotinylated secondary antibodies (1:1000) at roomtemperature overnight. The sections were washed and incubated inExtrAvidin™ (Sigma) (1:1000) for 3 hours and followed by H₂O₂ (0.01%) in3,3-diaminobenzine tetrahydrochloride (DAB, 0.05%) to produce a colouredreaction product. These sections were mounted on chrome alum-coatedslides, dried, dehydrated and covered.

The striatal neurons in both hemispheres exhibiting specificimmunoreactivities corresponding to ChAT were counted using a lightmicroscope and a 1 mm 2×1000 grid. The size of the striatal region usedfor the count was measured using an image analyser. The total counts ofneurons/mm² were compared between the groups.

Data were analysed using a paired t-test and presented as mean+/−SEM.Results are presented in FIG. 16.

Results

FIG. 16A shows that the number of ChAT-immunopositive neurons increasedin the brains of animals treated with G-2-MePE. This clearly indicatesthat administration of G-2-MePE is effective in increasing the level ofChAT in the brains of aged rats. Because ChAT is an enzyme involved inthe synthesis of the cholinergic neurotransmitter acetylcholine, weconclude that G-2-MePE can increase the amount of cholinergictransmitter in the brains of middle-aged rats.

Example 5 Effects of G-2-MePE on Spatial Reference Memory in Rats

Having demonstrated that G-2-MePE can increase ChAT and therefore hasthe potential to improve cholinergic neural function, we then examinedwhether G-2-MePE can be useful in treating age-related changes incognition and/or memory. Therefore, we carried out a series of studiesin rats using well-established tests for memory.

Experiment 1 The Morris Water Maze Test

The Morris water maze test is a well-recognized test to assess spatialreference memory in rats.

Subjects

We used male Wistar rats 12, 8 or 4 months of age.

Methods

Testing Environment and Apparatus

The Morris water maze test was conducted using a black plastic poolfilled to a depth of 25 cm with water colored black with a non-toxicdye. The pool had a circular black insert so that the walls alsoappeared uniform black The pool was divided into four quadrants (north,south, east and west) by two imaginary perpendicular lines crossing atthe pool's center A metal platform was placed in the geographical centreof the SE quadrant 50 cm from the edge of the pool, so that it was 2 cmbelow the water surface and invisible. The platform remained in thatposition though the training.

The experiment used extra-maze cues (i.e. objects in the roomsurrounding the pool) that the rats could use to navigate to theplatform. Distinctive posters or paintings were hung on the walls.Furniture in the room was not moved during the testing period. Theplacement of the pool allowed the experimenter an easy access to it fromall sides. The pool was emptied and refilled daily during testing, withwater at 25° C.+/−2° C.

The furthermost point in the pool (relative to the position of theexperimenter) was designated as “north”, and the other compass points“east”, “south” and “west” were the right-most, bottom and left-mostpoints of the pool respectively. These points were marked with tape onthe outside of the pool.

Acquisition Phase

Rats in each group were trained to swim to the submerged platform. Therats received six 60-second trials per day for four consecutive days. Atrial began by placing the rat into the water facing the wall of thepool, at one of four start locations (north, south, east, west). Thesequence of start locations was chosen pseudorandomly, so that the startlocation of any given trial was different from that of the previoustrial, and no start location was used more than twice during dailytraining. The same sequence of locations was used for all the rats on agiven day but varied between days. The trial ended when the rat hadfound the platform, or in 60 seconds, which ever occurred first. Thetrials were timed with a stop watch. If the rat found the platform, itwas allowed to remain there for 15 seconds before being removed to aholding container. If the platform was not found, the rat was guidedthere manually and placed on the platform for 15-seconds. Theinter-trial interval was 60 seconds. The holding container was coveredin order to minimize any inter-trial interference. At the completion ofdaily testing for a rat, the animal was towel-dried and placed under theheat lamp in the holding bucket until his coat was dry. The time neededto locate the platform (latency, sees) was obtained for each rat in eachtraining trial. If the rat did not find the platform in a given trialtheir latency score was the maximum length of that trial (60 seconds).

Drug Treatment

Three days after the completion of the acquisition phase, mini-osmoticpumps (Alzet) were implanted subcutaneously under halothane anesthesia)to dispense drug or vehicle continuously for 1 or 3 weeks. At thecompletion of the infusion the pumps were removed and the woundsre-sutured.

The 5 treatment groups were:

-   -   1. saline 1 week (n was originally 7, but one rat that lost        weight rapidly was excluded and later found to have had a        pituitary tumor);    -   2. saline 3 weeks (n=8);    -   3. G-2-MePE low dose (0.96 mg/day) 1 week (n=8);    -   4. G-2-MePE low dose (0.96 mg/day) 3 weeks (n=8);    -   5. G-2-MePE high dose (4.8 mg/day) 3 weeks (n=7).

The four (n=3) and eight month old (n=9) control rats received no drugtreatment. The 12-month old rats were assigned to one of five groups onthe basis of their swim times over acquisition, such that the groupswere approximately equivalent in their mean performance prior toreceiving any drug.

Retention (Reference Memory) Phase

The ability of the rats to remember or to relearn the original platformlocation was tested four weeks after original training. This means thatresidual drug would have been washed out for a minimum of 7 days in thecase of the 3-week pumps, and 21 days in the case of the 1-week pumps.The retention testing procedure was identical to that of acquisition.Pharmacokinetic studies indicate that the plasma concentration ofsubcutaneously administered G-2-MePE rose to a peak and then declinedwith an approximately first order kinetic pattern, with a plasmahalf-life (t 2) of between about 30 and 60 minutes. Thus, by the timethe retention study was performed, at least 7 days after removal of theG-2-MePE containing minipumps, nearly all of the G-2-MePE had beencleared from the animals' circulation.

Data Analysis

The swim latency for each rat was recorded for each trial for each dayof the acquisition and retention phases and changes between phases wereexamined using Analysis of Variance.

The 3-week vehicle and 3-week high dose G-2-MePE were compared inacquisition and retention. The high dose of G-2-MePE, given over 3 weeksimproved the retention of the original water maze task after a 4-weekdelay.

Results

FIG. 17 shows the comparison between high-dose (4.8 mg/day)G-2-MePE-treated and low-dose-treated (0.96 mg/day) aged rats and salinetreated aged rats, with the young controls (4 months) used as controls.Prior to treatment with G-2-MePE, there were no differences between theaged (12 month old) groups. In contrast, the 4 month old animalsrequired less time to reach the platform than older animals. After a3-week period of no testing, during which time either saline or G-2-MePEwere administered, animals that received saline only did not showimproved ability to reach the platform, as indicated by the similartimes required at test day 4 of the acquisition phase and test day 1 ofthe retention phase. In contrast, animals that received treatment withG-2-MePE at either the high or low doses, had improved memory asreflected in a decrease in the time needed to reach the platformcompared to saline-treated controls. Further, the G-2-MePE-treatedanimals had similar performance to the 4 month old young animals (FIG.17) and 8 month old animals (data not shown). Thus, we conclude thatG-2-MePE can improve memory in middle-aged rats animals that hadpreviously shown memory deficits in relation to young rats. Further,because by the time of retesting, the G-2-MePE had washed out from thecirculation, we conclude that the memory-enhancing effects of G-2-MePEwere likely due to the improvement in function of cholinergic neurons.

Experiment 2 8-Arm Radial Maze Test

Five months after the original experiment the now 17 month old rats wereretested for spatial working memory in a radial arm maze.

Methods

Apparatus

The apparatus consists of a central platform communicating with 8identical arms, each with a food cup at the end of the arm

Testing Procedure

Rats were partially food-deprived for at least 10 days prior to, andthroughout the radial maze procedure.

The maze was assembled and positioned so that the experimenter couldclearly observe the rats' behavior from a predetermined location. Theexperimenter numbered the arms of the maze according to theirorientation from one to eight in a clock-wise direction.

Pre-Training (Pre-Drug)

On day one the doors were inserted into the arms and each rat wasconfined in the central platform with 20 food pellets for 5 minutes.This continued once a day for four days, and all of the rats wereobserved to consume some of the pellets. The following day the rats wereallowed five minutes to explore the whole maze. All arms were baitedwith two food pellets in the food cup located at the end of each arm,and one pellet at both the entrance and middle of each arm. This wasrepeated for at least five, but up to eight days for rats that exploredfewer than eight arms in two consecutive sessions. All rats had a finalsession on the ninth day of pre-training. At this point it was decidedthat one of the old rats that had made only one arm entry on eight ofthe nine days should be excluded from future testing in this procedure.Otherwise all rats were included regardless of the amount of exploringthey performed in pre-training. There was no statistically significantdifference between the old groups in the number of arms entered on thefinal pre-training session (Drug: F(2,31)=0.44, p=0.65).

Drug Treatment

30 days before the test (five days after pre-training) the 17 MaleWistar month old rats were implanted (under halothane anesthesia) withsub-cutaneous mini-osmotic pumps (Alzet) to dispense drug continuouslyfor 3 weeks. At the completion of the infusion the pumps were removedand the wounds re-sutured (9-day washout allowed).

The treatment groups were:

1. young controls (4 months old), n=6;

2. saline n=10;

3. G-2-MePE low dose (2.4 mg/kg/day) n=13

4. G-2-MePE high dose (12.4 mg/kg/day) n=5

Saline and the low dose groups are comprised of all the rats thatreceived those treatments in phase 1 of this experiment (when the ratswere 12 months old) regardless of whether they had the one or three weektreatment. One rat in each of the saline and high dose groups have beendropped because of skin tumors. One of the low dose rats did notparticipate in this experiment due to the fact that it could not bepre-trained (see below).

Testing (Post-Drug)

Working memory testing commenced on the ninth day of washout. Ratsreceived 10 daily training sessions over 12 days. The procedure was thesame as for pre-training but only the food cups were baited. Rats had 6minutes to make up to 16 choices by visiting any of the eight arms. Achoice was defined as occurring when all four paws were inside an arm.The experimenter recorded the sequence of arm entries with pen andpaper, Sessions were terminated after all eight arms had been entered,16 choices made, or 6 minutes had elapsed. The time taken to enter alleight arms, when this occurred, was recorded.

Data Analysis

An arm choice was considered correct when the rat entered an arm notpreviously visited. Performance was classified daily according to thefollowing parameters:

1) Correct Choice (CC) 8-12 is the number of correct choices madedivided by the total number of choices made. For animals that failed tovisit all 8 arms in a test, the denominator of this ratio is consideredto be 12.

2) Working Correct Choice (WCC) 8-12 is the measure from which theworking memory data are derived. Data were collected as described for CC8-12 above, but for this parameter, only the rats that entered all 8arms in a session were included.

Rats that made fewer than 8 arm entries were not used to ascertainworking memory because they couldn't remember which arms they hadpreviously visited and therefore had memory so impaired that they couldnot complete the test, as opposed to the animals that, for whateverreason, did not explore the maze.

Results

CC8-12: There was a general improvement by all of the groups across the10 days (F(9,324)=4.01, p<0.0001), but no significant group effect(F(3,36)=1.19, ns) or Group X Days interaction (F(27,324)=1.05, ns)(data not shown)

WCC8-12: FIG. 18A shows the acquisition profile according to WCC8-12score across the 10 days of testing. There was a significant effect ofGroup (F(3,12)=4.27, p=0.029) and Days (F(9,108)=2.09, p=0.036) but theinteraction between these factors was not significant (F(27,108)=1.06,ns). The high dose G-2Me-PE group showed the greatest improvement acrossdays, followed by the young controls. There was very little differencebetween the low dose G-2Me-PE and saline.

FIG. 18B shows results indicating that rats exposed to the higher doseof G-2-MePE (n=5) had made more correct entries for getting food pelletscompared to the vehicle treated rats (*p<0.05, n=10). We conclude fromthis study that G-2-MePE improves spatial memory in aged rats.

Example 6 G-2-MePE Increases Neuroblast Proliferation and DecreasesAstrocytosis in Brains of Aged Rats

Because neuronal degeneration can result in decreased numbers ofneurons, one desirable therapeutic aim is increasing the numbers ofneurons in the brain. Neurons are derived from neuroblasts, a lessdifferentiated cell than a neuron, but within the neural lineage.Typically, a neuroblast is exposed to conditions that cause it to matureinto a mature phenotype, having a defined soma, neural processes (axonsand dendrites) and ultimately, making connections with other neurons(e.g., synapses). Thus, measuring neuroblast proliferation has become awell-known early marker for nerve cell proliferation. Thus, detecting anincrease in neuroblast proliferation induced by a pharmaceutical agentis an accepted method for predicting growth of neural cells in animals.Because rats and humans share similar mechanisms in neural cellproliferation, detection of changes in neuroblast proliferation in ratsin vivo is predictive of similar effects in human beings.

It is also known that one histological correlate of impaired cognitivefunction is an increase in the numbers of astrocytic cells in the brainof affected animals. Thus, to determine whether G-2-MePE might be usefulin stimulating neuroblast proliferation and in treating astrocytosis, wecarried out a series of studies in aging rats.

Methods and Materials

Immunohistochemistry

To carry out these studies, tissues were fixed and embedded in paraffinand sections obtained using standard methods. Coronal sections (6 μm)containing the level of the hippocampus were cut and mounted onchrome-alum-coated slides for staining. The sections were deparaffinizedin xylene, dehydrated in a series of ethanol and incubated in 0.1 Mphosphate buffered saline (PBS).

Primary antibodies against glial fibrillary acidic protein (GFAP) andproliferating cell nuclear antigen (PCNA) were used to mark reactiveglial cells and cells undergoing apoptosis and proliferation,respectively. For antigen unmasking (caspase-3 and PCNA staining),sections were heated in 10 mM sodium citrate buffer (pH 6.0) for 1 minat high power. All sections were pretreated with 1% H₂O in 50% methanolfor 30 min to quench the endogenous peroxidase activity. Then either1.5% normal horse serum or 2.5% normal sheep serum in PBS was appliedfor 1 h at room temperature to block nonspecific background staining.The sections were then incubated with following primary antibodies:monoclonal mouse anti-GFAP antibody (Sigma, St. Louis, Mo., U.S.A.diluted 1:500); mouse anti-PCNA antibody (DAKA, A/S, Denmark, diluted1:100). After incubation with primary antibodies at 4° C. for 2 d(except for PCNA staining which was incubated overnight) the sectionswere incubated with biotinylated horse anti-mouse or goat anti-rabbitsecondary antibody (1:200, Sigma) at 4° C. overnight. The ExtrAvidin™(Sigma, 1:200), which had been prepared 1 h before use, was applied for3 h at room temperature, and then reacted in 0.05% 3,3-diaminobenzidine(DAB) and PBS to produce a brown reaction product. Sections weredehydrated in a series of alcohols to xylene and coverslipped withmounting medium.

Immunohistochemical staining was performed on brain samples taken fromboth control and G-2-MePE treated groups of young (4 months old),middle-aged (9 months old) and aged rats (18 months old).

Control sections were processed in the same way except the primaryantibody was omitted from the incubation solution. The number of PCNApositive cells was counted in the subventricular zone and the GFAPpositive cells was scored in the cerebral cortex.

Experiment 1 G-2-MePE Stimulates Neuroblast Proliferation in Brains ofAged Rats

The subventricular zone (SVZ) and the dentate gyrus (DG) are two brainregions hosting adult neurogenesis. The reduction of neurogenesis inboth SVZ and the DG has been well reported to be co-related to thememory decline with aging and effects of Nerve Growth Factor andEpidermal Growth Factor on memory improvement are reported to be due toincrease in progenitors proliferation of the SVZ. Using PCNA as a markerof cell proliferation, cellular proliferation in the SVZ was examined bycounting the numbers of cells that are positive for PCNA. In selectedanimals, at least some of the proliferating cells were identified asneuroblasts, as stained with the neural-cell specific agent,doublecortin.

Eighteen month old male rats were treated intraperitoneally with singledoes of G2-MePE (doses of either 0, 0.012, 0.12. 1.2, 12 mg/kg). Brainswere collected 3 days after the treatments and the immunohistochemicalstaining of PCNA and GFAP were performed. The number of PCNA positivecells was counted in the SVZ and the number of cells was then averagedas cells/mm depending on the length of ventricle wall used for counting(FIG. 19A). The group treated with the highest dose (12 mg/kg, n=5)showed a significant increase in the number of PCNA positive cellscompared to the group treated with vehicle (*p<0.05, n=7). The dataindicated a dose-dependent effect of G-2PE on improving neurogenesis.

Fluorescence double labelling indicated co-localisation of PCNA withdoublecortin, a marker for neuroblasts. FIG. 19B is a photograph of aportion of a rat's brain showing an increase in both PCNA (green, ×20)and doublecortin (red, ×20) in the rat treated with the highest dose ofG-2-MePE (right panel) compared to the vehicle treated rat (left panel).The two markers clearly co-localised (FIG. 19B, photo, ×100). Weconclude that G-2-MePE can stimulate proliferation of brain cells,including neuroblasts. Because neuroblasts are precursor cells forneurons; we further conclude that G-2-MePE can increase the populationof neurons in the brains of animals treated with the compound of thisinvention.

Experiment 2 G-2-MePE Stimulates Neuroblast Proliferation in the SVZ ofBrains of Middle-Aged Rats

Effects of G-2-MePE (1.2 mg/kg) were studied in a group of middle-aged,9 month old rats. G-2-MePE (1.2 mg/kg) or vehicle was administeredintraperitoneally (i.p.). The proliferation of cells in the SVZ wasexamined 3 days after the treatment using PCNA immunohistochemicalstaining. FIG. 19C shows a significant increase in number of PCNApositive cells after the treatment of G-2-MePE (**p<0.005, n=4). Becausesome of the proliferating cells stained with PCNA were identified asneuroblasts (see Experiment 1 above), we conclude that G-2-MePE canstimulate neuroblast proliferation in middle-aged rat brains.

Experiment 3 Astrocytosis in Aging Brains

Growing evidence suggests that dysfunction of astrocytes in advanced agecan trigger inflammation, leading to further neuronral degeneration.Up-regulation of activated astrocytes has been well reported and isclosely associated with memory decline with aging, perhaps throughdepressed endogenous neurogenesis.

Using GFAP as a marker for reactive astrocytes, the number ofGFAP-positive cells was counted in the CA4 sub-region of the hippocampusof aged rats treated with G-2MeP or vehicle. We found a significantincrease in reactive astrocytes in the hippocampus of aged animals (FIG.20A), and in the cerebral cortex. Some of the astrocytes were associatedwith capillaries (FIG. 20B photo, arrows) in aged rats compared to bothyoung (*p<0.01) and middle aged rats (*#p<0.01).

As part of the vascular component, GFAP positive astrocytes also play arole in angiogenesis (FIG. 20B, arrows), which also contribute toinflammatory response in brains. Therefore the elevated GFAP astrocytesseen in aged brains may indicate a chronic stage of brain degeneration.

Experiment 4 G-2-MePE Reduces Astrocytosis in Aged Brains

We also evaluated effects of G-2-MePE on astrocytosis in the CA4sub-region of the hippocampus in aged rats. 18-month old male Wistarrats were assigned to 5 treatment groups as follows: vehicle, 0.12mg/kg/day, 0.12, 1.2 and 12 mg/kg/day (each n=6).

GFAP-positive cells were counted using a computerised program (Discovery1). Results are shown in FIGS. 20C and 20D. G-2-MePE was administeredintra-peritoneally and the numbers of GFAP-positive cells were assessed3d after the injection. Using a visual scoring system (0=no astrocytes,1=few astrocytes, 2<50%, 3>50%) we estimated the number of astrocytes in5 different cortical regions.

Treatment with G-2-MePE reduced number of reactive astrocytes in the CA4region of the hippocampus compared to the vehicle treated group (FIG.20C; *p<0.05), particularly the groups treated with doses of 0.12 and 12mg/kg. A similar effect was observed for G-2-MePE in the cerebral cortex(FIG. 20D).

Normally there are few GFAP-positive astrocytes located in the deeplayer of cortex of rat brains and those that are present are usually inclose association with white matter tracks. However, we have found therewere GFAP-positive cells in the middle layer of the cortex, closelyassociated with blood vessels.

Results of the studies presented herein indicate that aging isassociated with several changes in the brain. First, there is anage-dependent loss of memory and cognitive function, Second, there is anage-depended increase in astrocytes. All of these findings in the ratare consistent with each other and the known roles of cholinergic nervesin maintaining cognitive function and memory in experimental animals andin humans.

We unexpectedly found that a GPE analog, G-2-MePE, delivered to agedanimals at least partially reverses all of the above age-associatedchanges. First, G-2-MePE increases the amount of ChAT present in thebrain cells of animals exposed to the neurotoxins okadaic acid or 3-NP.This effect of G-2-MePE mimicked that of a well-known neuroprotectiveagent, GPE. These effects were seen in cortical cells, cerebellar cellsand in striatal cells, indicating that the effects were widespread indifferent portions of the brain. Second, G-2-MePE increased ChAT in thestriatum, indicating that cholinergic neurons are sensitive to G-2-MePE.These observed chemical and histological changes were paralleled bybehavioral changes. Aged animals treated with G-2-MePE exhibitedimproved memory in two well-known test systems compared tovehicle-treated controls. Next, G-2-MePE induced neuroblastproliferation in aging brains. Finally, treatment with G-2-MePE reversedthe increase in astrocytosis observed in the hippocampus and cortex ofaging brains. The effects of G-2-MePE were not due to acute effects ofthe agent; because in many of the studies cited herein, sufficient timehad elapsed from cessation of drug delivery to the test, that there waslikely little or no drug present.

Example 7 Comparison of the Pharmacokinetics of GPE and G-2-MePE

The purpose of these studies was to compare pharmacokinetic profiles ofGPE and G-2-MePE in animals in vivo using standard pharmacokineticmethods.

Methods

Adult male Wistar rats weighing between 180 and 240 g were used todetermine the pharmacokinetics of GPE and G2MePE. To facilitateintravenous bolus injections and blood sampling, all rats weresurgically implanted with an indwelling jugular venous cannula underhalothane anesthesia three days before the experiment. Groups of sixrats were given a single intravenous bolus injection of either 30 mg/kgGPE or 10 mg/kg G2MePE dissolved in 0.1M succinate buffer (pH 6.5).Blood samples (about 220 μl each) were collected into heparinized tubescontaining Sigma protease inhibitor cocktail for mammalian tissues at 10and 0 min before injection of either GPE or G2MePE, and 1, 2, 4, 8, 16,32, 64 and 128 min after injection of either GPE or G2MePE. The sampleswere centrifuged at 3000 g for 15 min at 4° C. and the plasma removedand stored at −80° C. until extraction and assay by eitherradioimmunoassay (“RIA”) or reverse phase HPLC. The RIA and HPLC methodsused were conventional.

Drug elimination after a single intravenous bolus injection was found tobe a first-order process following the equation C=C₀e^(−kt), where Crepresents drug concentration in any time point, C₀ is the concentrationwhen time (t) equals zero and k is the first-order rate constantexpressed in units of concentration per hour. The k and half-life(t_(1/2)) were calculated from the slope of the linear regression linein the elimination phase of the semi-logarithmic plot of plasmaconcentration versus time as: Log. C=−kt/2.3+log C₀. Results wereexpressed as mean±standard error.

Results

FIG. 21 shows a graph of plasma concentrations in vivo of GPE andG-2-MePE after intravenous (i.v.) injection. Filled squares representconcentrations of GPE at each time point, and filled triangles representconcentrations of G-2-MePE at each time point.

Plasma concentrations of GPE and G-2-MePE were markedly increased within1 min after injection. After injection of 30 mg/kg GPE, a peakconcentration of 40.0 t 10.8 mg/ml was observed. Plasma concentrationsof GPE then rapidly declined according to a first-order kinetic process.The first order rate constant for GPE was found to be 0.15±0.014ng/ml/min, the t_(1/2) was found to be 4.95±0.43 min and the estimatedclearance of GPE from plasma was found to be 137.5±12.3 ml/hr.

After injection of 10 mg/kg G-2-MePE, the peak concentration was foundto be 191±16.1 mg/ml. Plasma concentrations of G-2-MePE then declinedaccording to a first-order kinetic process. The first order rateconstant for G-2-MePE was found to be 0.033±0.001 ng/ml/min, the tin wasfound to be 20.7±0.35 min and the estimated clearance was found to be30.1±0.5 ml/hr.

After injection, the maximal plasma concentration of G-2-MePE was about4.8 times greater than the maximal plasma concentration of GPE, in spiteof the larger dose of GPE delivered (30 mg/kg) compared to the dose ofG-2-MePE delivered (10 mg/kg).

The finding of greater plasma concentrations of G-2-MePE than for GPE atall time points less than 125 minutes, in spite of a lower delivereddose of G-2-MePE, was totally unexpected based on known plasmaconcentrations of GPE. The t_(1/2) for G-2-MePE was over 4 times longerthan the tin for GPE.

The finding of increased half-life of G-2-MePE compared to that of GPEwas completely unexpected based on the t_(1/2) of GPE. The increasedt_(1/2) of G-2-MePE means that G-2-MePE is cleared more slowly from thecirculation. This finding is totally unexpected based on the clearancerate of GPE.

We conclude from these studies that G-2-MePE is a potent agent capableof reversing many of the adverse effects of aging in the brains ofanimals, including humans. GPE analogs, including G-2-MePE therefore,can produce desirable therapeutic effects, including neuroprotection,improved memory, increased neuroblast proliferation and reduction inastrocytosis, and can be valuable in reversing or mitigating adverseeffects of aging in humans.

While this invention has been described in terms of certain preferredembodiments, it will be apparent to a person of ordinary skill in theart having regard to that knowledge and this disclosure that equivalentsof the compound of this invention may be prepared and administered forthe conditions described in this application, and all such equivalentsare intended to be included within the claims of this application.

Example 8 Treatment of Rett Syndrome I Effects of G-2-MePE on Lifespanand Long-Term Potentiation in Rett Syndrome (RTT) Model

To determine whether G-2-MePE treatment can impact the development andprogression of Rett Syndrome in a murine model of the disorder, we usedhemizygous MeCP2(1lox) male mice. The MeCP2 knock-out (MeCP2-KO) mousesystem is widely accepted in the art as closely mimicking the range andthe severity of physiological and neurological abnormalitiescharacteristic of the human disorder, Rett Syndrome.

All experiments were performed at the University of Texas SouthwesternMedical Center and approved by the University of Texas SouthwesternMedical Center Animal Care and Use Committee. G-2-MePE was synthesisedAlbany Molecular Research Inc. (Albany, N.Y.) and supplied by NeurenPharmaceuticals Limited.

Methods

Treatment

We treated hemizygous MeCP2(1lox) male mice with 20 mg/kg/day ofG-2-MePE or saline, (0.01% BSA, n=15 per group in survival experimentand n=20 in the LTP experiment). The treatments were administeredintraperitoneally from 4 weeks after birth. For the survival experimentsthe treatment was maintained through the course of the experiment. Forthe LTP experiment the mice were treated until week 9 when they wereused for slice preparation.

Survival

MeCP2 deficient mutant mice develop RTT symptoms at about 4-6 weeks ofage and die between 10-12 weeks (Chen et al., 2001. Nat Genet 27:327-331). We compared the survival of the wild type controls and theMeCP2 deficient animals in vehicle- and G-2-MePE-treated groups.Survival was measured weekly from start of treatment (4 weeks) and usedto produce Kaplan-Meier survival curves to show the proportion of micethat survived (y axis) at each weekly interval (x axis) (see FIG. 22).

Long-Term Potentiation (Electrophysiology)

MeCP2 deficient mice have been previously reported to suffer fromfunctional and ultrastructural synaptic dysfunction, significantimpairment of hippocampus-dependent memory and hippocampal long-termpotentiation (LTP) (Moretti et al. The Journal of Neuroscience. 2006.26(1):319-327). To test the effects of the G-2-MePE treatment onsynaptic function in the RTT model we compared hippocampal LTP in bothvehicle and G-2-MePE treated animals at 9 weeks of age. To do so, wemeasured the slope of the fEPSP as a % of baseline potential in neuronsin slices of hippocampus from MeCP2 deficient mice treated with eithersaline or G-2-MePE (FIG. 23).

Results

FIG. 22 shows that G-2-MePE treatment increased survival of MeCP2deficient mice. Wild-type mice (top line) are control animals, andtherefore their survival was 100% at each time point. MeCP2 deficientmice treated with saline only died much more rapidly (dotted line) thanwild-type mice, such that by about 11 weeks, only 50% of the MeCPdeficient mice survived. In striking contrast, however, we unexpectedlyfound that MeCP2 deficient mice treated with G-2-MePE survivedsubstantially longer than saline-treated mice. At about 15 weeks, 50% ofthe animals survived. Data initially presented showed that MeCP2 micewere impacted in terms of survival such that 50 percent of animals haddied by 11 weeks in the untreated case. G-2-MePE treated animals showedimproved survival, with 50 percent having died at 16 weeks. In thisstudy, the longevity data were compromised by inconsistent veterinaryprocedures, such that mice did not have their teeth clippedconsistently—a requirement in mecp2 mice unrecognized at the start ofthe experiment. A consequence was the observation of early animal deathsunrelated to Rett Syndrome (particularly in the control group).Re-examination of the data showed that the effect of G-2-MePE persistedwhen the control group was re-run, albeit the difference in groups beingsmaller (time to 50 percent death 13.5 weeks in controls, 16 weeks inG-2-MePE treated animals). No safety concerns were raised by G-2-MePEtreatment of mecp2 mice.

These results demonstrated that G-2-MePE can substantially increasesurvival of MeCP2 deficient mice. Because MeCP2 deficient mice arepredictive of the pathology and therapeutic efficacy in human beingswith Rett Syndrome, we conclude that G-2-MePE can increase life span ofhuman beings with Rett Syndrome.

FIG. 23 shows results of our studies to determine if G-2-MePE treatmentincreased hippocampal long-term potentiation (LTP) as measured by thefEPSP slope in MeCP2 deficient animals compared to saline-treated mutantmice. As shown in FIG. 23, we unexpectedly found that G-2-MePE increasedthe slope of fESPS in MeCP2 deficient mice compared to animals treatedwith saline only.

These results demonstrated that G-2-MePE can be effective in treatingMeCP2 deficient mice in vivo. Because MeCP2 deficient mice arepredictive of the pathology and therapeutic efficacy in human beingswith Rett Syndrome, we conclude that G-2-MePE can be an effectivetherapy for people with Rett Syndrome.

Example 9 G-2-MePE Improves Dendritic Arborization and IncreasesDendritic Spine Length

We assess the effects of G-2-MePE treatment on dendrites. Transgenicmecp2 knockout mice (n=15 to 20) were administered G-2-MePEintraperitoneally at a dose of 20 mg/kg once daily. Following sacrificedendritic spine density, spine length and aborization were examinedafter Golgi staining after nine weeks, as per the Table 1 below:

TABLE 1 Sample size for all neuron morphologic and spine analysis MALESample size (average Sample size number of neurons or (no. of mice)dendrites per animal) AGE KO- KO-NNZ- KO- KO-NNZ- Analysis (Weeks)vehicle G-2MePE vehicle G-2MePE Neuron 9 3 3 4 4 morphology SpineAnalysis 9 3 3 10 10Dendritic length was assessed by distance from the soma ofrepresentative hippocampal CA1 neurons from 9 week old male mecp2 nullmutant mice treated with either saline (3 neurons analysed from 3separate mice, n=9) or G-2-MePE (20 mg/kg i.p. 1/day, from week 4; 3neurons analysed from 3 separate mice, n=9).

We observed that G-2-MePE improved dendritic arborization and increaseddendritic spine length. FIG. 24 depicts results of this study. Dendriticlength in μm (vertical axis) is plotted against the distance (in μm;horizontal axis) from the soma of the cells. For cells with dendritesclose to the somas, the dendrites were short. However, as the distancefrom the somas increased saline-treatment (open squares) produceddendritic lengths that increased to a maximum at a distance of 70 μmfrom the soma and declined at distances further away from the somas. Incontrast, treatment with G-2MePE (filled squares) produced longerdendrites over much of the range of distances from the somas.

Example 10 Treatment of Rett Syndrome in Mice II Mice Mating andGenotyping

The MeCP2 germline null allele mice are used (Chen et al., 2001).Genotyping is performed as in Chen et al. (Chen et al., 2001).

G-2-MePE Treatment

For the survival measurements, the nocturnal activity analysis and theimmunoblot analysis, G-2-MePE (synthesised Albany Molecular ResearchInc. (Albany, N.Y.) and supplied by Neuren Pharmaceuticals Limited) isadministered daily via intra-peritoneal injections (20 mg/kg,vehicle=saline, 0.01% BSA). The treatment starts at PI 5 and ismaintained throughout the course of the experiments. For intracellularphysiology experiments, the mice are injected daily with G-2-MePE (20mg/kg body weight, vehicle=saline, 0.01% BSA) for 2 weeks, from P15 toP28-P32 when they are used for acute slice preparation. For opticalimaging experiments, mice are injected with G-2-MePE (20 mg/kg bodyweight, vehicle=saline, 0.01% BSA) daily from the day of the lid sutureto the day of imaging.

Slice Physiology Preparation

Coronal sections (300 μm thick) at or near sensorimotor cortex are cutin <4° C. ACSF using a Vibratome. Slices are incubated at 37° C. for 20minutes after slicing, and at room temperature for the remainder of theexperiment. Slices are transferred to a Warner chamber and recordingsare taken from visually identified pyramidal neurons located in layer 5.Artificial cerebral spinal fluid (ACSF) containing 126 mM NaCl, 25 mMNaHCO3, 1 mM NaHPO4, 3 mM KCl, 2 mM MgSO4, 2 mM CaCl2, and 14 mMdextrose, is adjusted to 315-320 mOsm and 7.4 pH, and bubbled with 95%O2/5% CO2. The intracellular pipette solution contained 100 mM potassiumgluconate, 20 mM KCl, 10 mM HEPES, 4 mM MgATP, 0.3 mM NaGTP, and 10 mMNa-phosphocreatine.

Intracellular Whole-Cell Recordings

Borosilicate pipettes (3-5 MΩ, WP1) are pulled using a Sutter P-80puller (Sutter Instruments). Cells are visualized with an Achroplan 40×water-immersion lens with infrared-DIC optics (Zeiss) and detected withan infrared camera (Hamamatsu) projecting to a video monitor.Experiments are driven by custom acquisition and real-time analysissoftware written in Matlab (Mathworks, Natick, Mass.) using a Multiclamp700B amplifier (Axon Instruments) connected to a BNC-2110 connectorblock and M-Series dual-channel acquisition card (National Instruments).Gigaseal and rupture is achieved and whole-cell recordings arecontinuously verified for low levels of leak and series resistance. Foreach recording, a 5 mV test pulse is applied in voltage clamp ˜10 timesto measure input and series resistance. Then in current clamp ˜10 pulses(500 ms, 40-140 pA at 10 pA increments), are applied to quantify evokedfiring rates and cellular excitability. Access resistance, leak, andcellular intrinsic excitability are verified to be consistent acrossgroups. Finally, spontaneous EPSCs under voltage clamp at −60 mV aresampled at 10 kHz and low-pass filtered at 1 kHz. Analysis is performedusing a custom software package written in Matlab, with all eventsdetected according to automated thresholds and blindly verified for eachevent individually by the experimenter.

Golgi Staining

Samples (<1 cm) from P28 mice are fixed in 10% formalin and 3% potassiumbichromate for 24 hours. Tissue is then transferred into 2% silvernitrate for 2 days in the dark at room temperature. Sections from thesesamples are then cut at 50 μm thickness into distilled water. Sectionscorresponding to motor cortex are mounted onto slides, air dried for 10minutes, and then dehydrated through sequential rinses of 95% alcohol,100% alcohol, and xylene, and then sealed with a coverslip. Images reacquired at 10× (whole cell) and 100× (spine imaging) using a ZeissPascal 5 Exciter confocal microscope.

Optical Imaging of Intrinsic Signals

Adult (>P60) wild type (SVEV or BL6) and MeCP2 (+/−) mutant females(BL6) are used for this experiment. The wild type control group iscomposed of both wild type littermates of MeCP2+/− females or wild typeage matched SVEV females. For monocular deprivation, animals areanesthetized with Avertin (0.016 ml/g) and the eyelids of one eye issutured for 4 days. Prior to imaging, the suture is removed and thedeprived eye re-opened. Only animals in which the deprivation suturesare intact and the condition of the deprived eye appears healthy areused for the imaging session. For G-2-MePE signaling activation, asolution containing G-2-MePE is injected intra-peritoneally (IP) dailyfor the entire period of deprivation. For the imaging sessions mice areanesthetized with urethane (1.5 g/kg; 20% of the full dosage isadministered IP each 20-30 minutes up to the final dosage, 0.02 ml ofcloroprothixene 1% is also injected together with the firstadministration). The skull is exposed and a custom-made plate is gluedon the head to minimize movement. The skull is thinned over V1 with adremel drill and covered with an agarose solution in saline (1.5%) and aglass coverslip. During the imaging session, the animal is constantlyoxygenated, its temperature maintained with a heating blanket and theeyes periodically treated with silicone oil; physiological conditionsare constantly monitored. The anesthetized mouse is placed in front of amonitor displaying a periodic stimulus presented to either eye,monocularly; the stimulus consisted of a drifting vertical or horizontalwhite bar of dimensions 9°×72°, drifting at 9 sec/cycle, over auniformly gray background. The skull surface is illuminated with a redlight (630 nm) and the change of luminance is captured by a CCD camera(Cascade 512B, Roper Scientific) at the rate of 15 frames/sec duringeach stimulus session of 25 minutes. A temporal high pass filter (135frames) is employed to remove the slow signal noise, after which thesignal is computer processed in order to extract, at each pixel, thetemporal Fast Fourier Transform (FFT) component corresponding to thestimulus frequency. The FFT amplitude is used to measure the strength ofthe visual evoked response to each eye. The ocular dominance index isderived from each eye's response (R) at each pixel asODI=(Rcontra−Ripsi)/(Rcontra+Ripsi). The binocular zone is defined asthe region activated by the stimulation of the eye ipsilateral to theimaged hemisphere.

Heart Rate Measurements

Real time cardiac pulse rate is measured using a tail clip sensor (MouseOX Oximeter—Oakmont, Pa.). Mice are not anesthetized but physicallyrestrained in a fitted open plastic tube. Prior to the recording sessionthe tube is placed overnight in the cages housing the experimentalanimals to allow habituation. Body temperature is maintained at ˜82-84°F. throughout the recording time. We record 3 trials of 15 minutes foreach mouse, mice are 8 weeks old and treated with vehicle or G-2-MePEfrom P15.

Nocturnal Activity Measurements

Spontaneous motor activity is measured by using an infraredbeam-activated movement-monitoring chamber (Opto-Varimax-MiniA; ColumbusInstruments, Columbus, Ohio). For each experiment, a mouse is placed inthe chamber at least 3 h before recordings started. Movement ismonitored during the normal 12-h dark cycle (7 p.m. to 7 a.m.). One darkcycle per animal per time point is collected.

Results

To test whether G-2-MePE treatment will impact the development ofcardinal features of the RTT disease, 2 week old mutant animals aregiven daily intra-peritoneal injections for the course of theirlifespan. Measurements of synaptic physiology, synaptic molecularcomposition, and cortical plasticity are then acquired as detailedbelow, along with health-related measurements such as heart rate,locomotor activity levels, and lifespan.

Effects of G-2-MePE on the Synaptic Physiology of MeCP2 Mutant Mice

Recent studies have reported that neurons across multiple brain regionsof MeCP2−/y mice display a profound reduction in spontaneous activity(Chang et al., 2006; Chao et al., 2007; Dani et al., 2005; Nelson etal., 2006) a phenotype that is rescued by over-expression of BDNF (Changet al., 2006). Similarly, acute application of an IGF1 derivative hasbeen shown to elevate evoked excitatory postsynaptic current (EPSC)amplitudes by 40% in rat hippocampal cultures (Ramsey et al., 2005; Xinget al., 2007). To test the efficacy of G-2-MePE in rescuing the MeCP2−/yphysiological phenotype, we acquire intracellular whole cell recordingsin acute brain slices, measuring excitatory synaptic drive (spontaneousEPSC amplitude and frequency) in layer 5 cortical neurons. Here, EPSCsrecorded from −/y animals are significantly reduced in amplitudecompared to EPSCs measured in wild-type animals. The trend is partiallyreversed in EPSCs recorded from MeCP2−/y animals treated with G-2-MePE,which are significantly larger in amplitude than EPSCs from MeCP2−/ymice treated with vehicle. These differences are also seen whenaveraging across cells. Throughout these measurements, accessresistance, leak, and cellular intrinsic excitability are also verifiedto be consistent across groups. Quantifying EPSC intervals also shows aslight increase in the interval between EPSC events (reduced EPSCfrequency) between wild-type and MeCP2−/y animals (P=0.04,Kolmogorov-Smirnov test). Our findings thus indicate that the reductionof excitatory synaptic drive in cortical cells of MeCP2−/y mice, and itspartial rescue following G-2-MePE treatment, are due in part to a changein EPSC amplitude as a consequence of a change in the strength of thesynapses mediating excitatory transmission in this region.

G-2-MePE Treatment Stimulates Cortical Spine Maturation

We use Golgi staining to label neurons sparsely and distinctly, andapplied high-resolution confocal imaging to measure dendritic spinedensity and morphology in the labelled cells, restricting analysis tolayer 5 pyramidal neurons in sections of motor cortex from criticalperiod mice (P28).

While low-magnification imaging clearly delineates the extent of thedendrites of the pyramidal cells we use higher magnifications to countsynaptic contacts and determine the morphological class of each spine.We classify spines as either large and bulbous (“mushroom”, M), shortand stubby (“stubby”, S), short and thin (“thin”, T) or filopodia (F).Comparing the density of spines per unit branch exhibits a trend ofdecreased spine density in knockout neurons that is largely amelioratedin the knockout with treatment.

Together these results indicate the potential for deficits in the numberand maturational status of dendritic contacts in the knockout tounderpin functional defects in excitatory transmission, in a manner thatcan be treated following administration of G-2-MePE.

Ocular Dominance (OD) Plasticity in Adult MeCP2+/−Mice is Reduced byG-2-MePE

Developmental changes in OD plasticity are controlled in part by theactivation of the IGF-1 pathway, and administration of (1-3)IGF-1 canreduce OD plasticity in wild type young mice (Tropea et al., 2006). Wetherefore test if G-2-MePE treatment could stabilize the prolonged ODplasticity observed in adult MeCP2 mutants. Female MeCP2+/− mice, agedP60 or more, are monocularly deprived for 4 days and treatedconcurrently with G-2-MePE. G-2-MePE treatment reduces the OD plasticityin the adult Mecp2+/− mice, indicating that indeed G-2-MePE can rapidlyinduce synapse stabilization or maturation.

Bradycardia in MeCP2−/y Mice is Treated by G-2-MePE

In addition to examining the efficacy of G-2-MePE in amelioratingneurophysiological symptoms, we seek to characterize its effects on thegeneral health of the organism. Clinical and experimental evidence showsautonomic system dysfunctions such as labile breathing rhythms andreduced baseline cardiac vagal tone in Rett Syndrome patients (Julu etal., 2001). A poor control of the feedback mechanisms that regulateblood pressure homeostasis through the sympathetic system, for examplehyperventilation-induced decrease in heart rate, is common in RettSyndrome patients and can cause life threatening cardiac arrhythmias(Acampa and Guideri, 2006; Julu et al., 2001).

The pathogenesis of the cardiac dysautonomia, although not wellunderstood, suggests that immature neuronal connections in the brainstemcould be the cause. To examine heart rate abnormalities in MeCP2−/y miceand the effect of G-2-MePE treatment, we monitor real time cardiac pulserate in non-anesthetized wild type and MeCP2−/y animals treated withvehicle or G-2-MePE. Wild type mice exhibit a regular distribution ofheart rate measurements centred near 750 beats per minute. In contrast,MeCP2−/y mice exhibit a more irregular heart rate with a lower averagerate, the occurrence of which is significantly reduced followingtreatment with G-2-MePE.

G-2-MePE Administration Improves Locomotor Activity and Life Span

MeCP2−/y mice develop Rett-like symptoms beginning at 4-6 weeks of agewhen they progressively become lethargic, develop gait ataxia and diebetween 10 and 12 weeks of age (Chen et al., 2001). Baseline locomotoractivity is also recorded in mice after 6 weeks by counting nocturnalinfrared beam crossing events within a caged area. MeCP2 knockout mice(KO) exhibits markedly reduced locomotor activity levels compared towild-type mice (WT), but treatment with G-2-MePE (KO-T) elevates theselevels.

Finally, compared to MeCP2 KO littermates, MeCP2−/y mice treated withG-2-MePE also show a ˜50% increase in life expectancy (an increase inthe 0.5 probability survival rate).

We also measure the effect of G-2-MePE treatment on neuron soma size inthe hippocampus. Mice are treated with G-2-MePE as described above forlocomotor activity. Soma size in neurons in the CA3 region of thehippocampus is significantly impaired in MeCP2 KO animals relative towild-type animals. G-2-MePE treatment increases average soma size in KOanimals, but has little or no effect on soma size in wild type animals.

Example 11 Effect of Oral G-2-MePE on Survival in Rett Syndrome in Mice

Because Rett Syndrome is a chronic, debilitating disorder involving lossof motor skills, it is desirable to treat Rett Syndrome using easilyadministered preparations. To this end, we can take advantage ofunexpectedly beneficial therapeutic and pharmacokinetic properties ofG-2-MePE and related compounds (U.S. Pat. Nos. 7,041,314, 7,605,177,7,714,070, 7,863,309 and U.S. application Ser. Nos. 11/315,784 and12/903,844).

Therefore, we administer G-2-MePE orally to MeCP2 deficient mice asdescribed in US 2009/0074865. Briefly, an aqueous solution, awater-in-oil emulsion (micro-emulsion, coarse emulsion or liquidcrystal), or a gel composition containing a pharmaceutically effectiveamount of G-2-MePE (20 or 80 mg/kg per animal) is administered daily. Incontrol MeCP2 deficient animals, we administer saline only, andwild-type animals are used to obtain baseline data similar to the designof studies described in Example 8 above.

In wild-type animals, survival is defined to be 100% at each time point.In MeCP2 deficient animals, survival is decreased substantially.However, after oral administration of G-2-MePE to MeCP2 deficient mice,survival is increased substantially.

Example 12 Effect of G-2MePE on Seizure Activity in Rett Syndrome inMice

Because seizures are a prominent, hazardous and a difficult to treataspect of Rett Syndrome, we determine the effects of G-2MePE on seizureactivity in MeCP2 deficient animals. G-2-MePE can be effective intreating seizure activity in animals with neurodegenerative disease(U.S. Pat. No. 7,714,020). Therefore, we carry out experiments todetermine whether G-2-MePE can also treat seizure activity in MeCP2deficient mice.

Electroencephalograpic recordings of wild-type mice and MeCP2 deficientmice treated with either saline or G-2-MePE are obtained using methodsdescribed in U.S. Pat. No. 7,714,020.

We find that G-2MePE can be effective in decreasing both motor seizuresand non-convulsive seizures.

Conclusions

Based on our in vivo and in vitro studies in MeCP2 deficient animals, weconclude that G-2-MePE can be an effective therapy for treating humanbeings with Rett Syndrome. Moreover, because G-2-MePE has unexpectedlylonger half life than a naturally occurring compound ((1-3) IGF-1;Glycyl-Prolyl-Glutamate or GPE) (FIG. 21), we conclude that use ofG-2-MePE has distinct and substantial advantages over otherpharmacological agents, including GPE.

For example, G-2-MePE is not degraded by gastrointestinal cells, istaken up by gastrointestinal cells, and is active in the central nervoussystem after oral administration (Wen et al., U.S. application Ser. No.12/283,684; U.S. 2009/0074865, U.S. Pat. No. 7,887,839, incorporatedherein fully by reference), Therefore, G-2MePE need not be deliveredintravenously, subcutaneously, intraventricularly, or parenterally. Infact, oral formulations comprising micro-emulsions, coarse emulsions,liquid crystal preparations, nanocapsules and hydrogels can be used inmanufacture of orally administered preparations such as tablets,capsules and gels that can improve neurological function and treatneurodegenerative conditions (U.S. Pat. No. 7,887,839). Compounds ofthis invention can be used in situations in which a patient's motorfunctioning is below that needed to swallow a table or capsule. Thereare several types of soluble gels for oral administration of compounds,and these can be used to deliver a compound or composition of thisinvention to a patient. Because G-2-MePE can be easily administeredorally and is orally effective in treating neurodegenerative disorders,including Rett Syndrome, we conclude that G-2-MePE can be convenient andbeneficial for long-term therapy of patients with Rett Syndrome.

Further, because Rett Syndrome shares key features with other autismspectrum disorders, compounds of this invention can be useful inproviding therapeutic benefit from animals having other ASD, and inhumans with autism, Asperger Syndrome, Childhood DisintegrativeDisorder, and Pervasive Developmental Disorder—Not Otherwise Specified(PDD-NOS).

Example 13 Treatment of ASD

Shank3-Deficient Mouse Model

Shank3-deficient mice are used in the study as a model of 22q13 deletionsyndrome associated with ASD.

22q13 deletion syndrome has been linked with deletions or mutations inShank3 gene (Bonaglia et al, 2006). The Shank3 gene codes for a masterscaffolding protein which forms the framework in glutamatergic synapses(Boeckers et. al, 2006). Shank3 is a crucial part of the core of thepostsynaptic density (PSD) and recruits many key functional elements tothe PSD and to the synapse, including components of theα-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA);metabotropic glutamate (mGlu), and N-methyl-D-aspartic acid (NMDA)glutamate receptors, as well as cytoskeletal elements. Recent studiesexploring the rate of 22q13 deletions/Shank3 mutations suggest thathaploinsufficiency of Shank3 can cause a monogenic form of ASD with afrequency of 0.5% to 1% of ASD cases (Durand et al, 2007; Moessner etal, 2007; Gauthier et al, 2008).

The generation of the mouse model with disrupted expression offull-length Shank3 has been previously described in the art (Bozdagi etal., Molecular Autism 2010, 1:15, p 4). Briefly, Bruce4 C57BL/6embryonic stem cells were used to generate a mouse line that had loxPsites inserted before exon 4 and exon 9. The floxed allele was excisedand a line was maintained with a deletion of exons 4 to 9, i.e. acomplete deletion of the ankyrin repeat domains of Shank3. Wild-type(+/+), heterozygous (+/−) and knockout (−/−) mice were produced, withMendelian frequencies from heterozygote-heterozygote crosses. A 50%reduction of full length Shank3 mRNA was confirmed in heterozygotes(qPCR) as well as a reduced expression of Shank3 protein (byimmunoblotting with Shank3 antibody N69/46).

Heterozygous mice generated by crossing wild-type mice withheterozygotes are used in this example to best model thehaploinsufficiency of Shank3, responsible for 22q13 deletion syndrome.

Methods

Drug Treatment

1 to 3 month old wild type and heterozygous Shank3-deficient mice aredivided into 4 treatment groups: placebo treated wild-type, placebotreated Shank3-deficient group and two Shank3-deficient G-2-MePE treatedgroups. The animals are given placebo (water) or G-2-MePE formulated inwater administered orally, b.i.d for 14 days. G-2-MePE is administeredat two doses: 15 or 60 mg/kg.

Methodology

A detailed description of the methodology can be found in Bozdagi et al.(Molecular Autism 2010, 1:15).

Behavioral Analyses

Behavioral assessments are made at several, time points, and includeanalysis of social interactions and ultrasonic social communication, inline with the methodology described by Bozdagi et al. Briefly,male-female social interactions in each treatment group are evaluated.The subject males are group-housed and individually tested in cleancages with clean litter. Each testing session lasts 5 min. Each of thesubject mice is paired with a different unfamiliar estrus C57BL/6Jfemale. A digital closed circuit television camera (Panasonic, Secaucus,N.J., USA) is positioned horizontally 30 cm from the cage. An ultrasonicmicrophone (Avisoft UltraSoundGate condenser microphone capsule CM15;Avisoft Bioacoustics, Berlin, Germany) is mounted 20 cm above the cage.Sampling frequency for the microphone is 250 kHz, and the resolution is16 bits. While the equipment used cannot distinguish between callsemitted by the male subject and female partner, the preponderance ofcalls during male-female interactions in mice is usually emitted by themale. The entire apparatus is contained in a sound-attenuatingenvironmental chamber (ENV-018V; Med Associates, St Albans, Vt., USA)illuminated by a single 25-Watt red light. Videos from the male subjectsare subsequently scored by an investigator uninformed of the subject'sgenotype and treatment group on measures of nose-to-nose sniffing,nose-to-anogenital sniffing and sniffing of other body regions, usingNoldus Observer software (Noldus Information Technology, Leesburg, Va.,USA). Ultrasonic vocalizations are identified manually by two highlytrained investigators blinded to genotype/treatment group information,and summary statistics are calculated using the Avisoft package.Interrater reliability is 95%. Data are analysed using an unpairedStudent's t-test.

Olfactory habituation/dishabituation testing is conducted in male andfemale mice for each group. The methodology is as previously described(Silverman et al 2010, Yang et al 2009 and Silverman et al 2010).Non-social and social odors are presented on a series of cotton swabsinserted into the home cage sequentially, each for 2 min, in thefollowing order: water, water, water (distilled water); almond, almond,almond (1:100 dilution almond extract); banana, banana, banana (1:100dilution artificial banana flavouring); social 1, social 1, social 1(swiped from the bottom of a cage housing unfamiliar sex-matched B6mice); and social 2, social 2, social 2 (swiped from the bottom of asecond cage housing a different group of unfamiliar sex-matched129/SvImJ mice). One-way repeated measures ANOVA is performed withineach treatment group for each set of habituation events and eachdishabituation event, followed by a Tukey post hoc test.

Hippocampal Slice Electrophysiology

Post-mortem, acute hippocampal slices (350 μm) are prepared from miceusing a tissue chopper. Slices are maintained and experiments areconducted at 32° C. Slices are perfused with Ringer's solutioncontaining (in mM): NaCl, 125.0; KCl, 2.5; MgSO₄, 1.3; NaH₂PO₄, 1.0;NaHCO₃, 26.2; CaCl₂, 2.5; glucose, 11.0. The Ringer's solution isbubbled with 95% O2/5% CO2, at 32° C., during extracellular recordings(electrode solution: 3 M NaCl). Slices are maintained for 1 hr prior toestablishment of a baseline of field excitatory postsynaptic potentials(fEPSPs) recorded from stratum radiatum in area CA1, evoked bystimulation of the Schaffer collateral-commissural afferents (100 μspulses every 30 s) with bipolar tungsten electrodes placed into areaCA3. Test stimulus intensity is adjusted to obtain fEPSPs withamplitudes that are one-half of the maximal response. The EPSP initialslope (mV/ms) is determined from the average waveform of fourconsecutive responses. Input-output (I/O) curves are generated byplotting the fEPSP slope versus fiber volley amplitude in low-Mg²⁺ (0.1mM) solution. AMPA receptor-mediated and NMDA receptor-mediated I/Orelationships are measured in the presence of ionotropic glutamatereceptor antagonists: 2-amino-2-phosphonopentanoic acid APV (50 μM) and6-cyano-7-nitroquinoxaline-2,3-dione CNQX (100 μM). Paired-pulseresponses are measured with interstimulus intervals of 10 to 200 ms, andare expressed as the ratio of the average responses to the secondstimulation pulse to the first stimulation pulse.

LTP is induced either by a high-frequency stimulus (four trains of 100Hz, 1 s stimulation separated by 5 min), or by theta-burst stimulation(TBS) (10 bursts of four pulses at 100 Hz separated by 200 ms), or by asingle 100 Hz stimulation, for control and genetically-modified mice. Toinduce long-term depression (LTD), Schaffer collaterals are stimulatedby a low frequency or paired-pulse low frequency stimulus (900 pulses at1 Hz for 15 min) to induce mGlu receptor-dependent LTD. Data areexpressed as means±SD, and statistical analyses are performed usinganalysis of variance (ANOVA) or student's t-test, with significance setat an a level of 0.05.

Results

Behavioral

Cumulative duration of total social sniffing by the male test subjectsis lower in placebo treated Shank3-deficient group than in placebotreated wild-type group. In addition, fewer ultrasonic vocalizations areemitted by the placebo treated Shank3-deficient group than by thewild-type controls during the male-female social interactions.

G-2-MePE treatment in the two Shank3-deficient groups results in asignificant increase in the cumulative duration of total social sniffingin comparison to the placebo treated Shank3-deficient group. Moreover,the G-2-MePE treated groups display an increased number of ultrasonicvocalizations than the placebo treated mutant group.

In the olfactory habituation/dishabituation study, intended to confirmthat the mice are able to detect social pheromones, all 4 groups displaynormal levels of habituation (indicated by decreased time spent insniffing the sequence of three same odors), and the expecteddishabituation (indicated by increased spent in sniffing the differentodor).

Electrophysiology

Plotting field excitatory postsynaptic potential (fEPSP) slope versusstimulus intensity demonstrates a reduction in the I/O curves in theplacebo treated Shank3-deficient group versus the control group. In theheterozygous placebo treated group we also observe a decrease in AMPAreceptor-mediated field potentials, reflected in a 50% decrease in theaverage slope of I/O function compared to the wild-type control group.In contrast, when the I/O relationship is analysed in the presence ofthe competitive AMPA/kainate receptor antagonist CNQX to measuresynaptic NMDA receptor function, there is no difference between thewild-type and placebo treated heterozygous groups. These resultsindicate that there is a specific reduction in AMPA receptor-mediatedbasal transmission in the Shank3 heterozygous mice.

G-2-MePE treatment in both heterozygous groups normalizes the AMPAreceptor-mediated field potentials and causes an increase in the averageslope of I/O function compared to the placebo treated Shank3-deficientgroup.

The maintenance of LTP in the placebo treated Shank3-deficient group isclearly impaired in comparison to the wild-type control. TBS LTP tests(10 bursts of four pulses at 100 Hz separated by 200 ms) also show asignificant decrease in the potentiation at 60 min after TBS in theplacebo treated Shank3-deficient group. In contrast to the alteredsynaptic plasticity observed with LTP, long-term depression (LTD) wasnot significantly changed in the mutant group. G-2-MePE treatmentincreased hippocampal long-term potentiation (LTP) and its maintenancein both Shank3-deficient group in comparison to the placebo treatedShank3-deficient group.

Discussion

Poor social competencies and repetitive behaviors are the commonfeatures and key diagnostic measures of all forms of ASD, Delayedintellectual development and underdeveloped language skills are also acommon feature present in all ASD, excluding Asperger syndrome.

The animal models described above have been accepted in the art asdemonstrating similar symptoms to the clinical human conditions. Allmutant models discussed above (NLGN3, NLGN4, CADM1, NRXN1, FMR1, shank3)exhibit impaired social skills or increased social anxiety. Decreasedexcitatory transmission into the hippocampus has been identified inNRXN1, shank3, MeCP2 and FMR1 mutant animal models. At present nopolygenetic or multifactorial models of ASD have been described. Theanimal models described above, based on genetic defects that are knownto produce ASD in human population, provide the best opportunity to testthe efficacy of ASD therapies.

Therefore the efficacy of G-2-MePE in animal models of ASD is reasonablypredictive of its efficacy in a human subject suffering from ASD.

Example 14 G-2-MePE Treatment Changes the Morphology of Neurons in an InVitro Human Model of Rett Syndrome

To test the effects of G-2-MePE on neuronal morphology, we used an invitro model of RTT described in Marchetto et al., A model for neuraldevelopment and treatment of Rett syndrome using human inducedpluripotent stem cells, Cell 143:527-539 (2010) (including supplementalinformation). The model uses induced pluripotent stem cells (iPSCs)generated from fibroblasts of human RTT patients carrying differentMeCP2 mutations.

Methods

Cell Culture and Retrovirus Infection

RTT fibroblasts (carrying 4 distinct MeCP2 mutations) and controlfibroblasts are generated from explants of dermal biopsies. The shRNAagainst target MeCP2 gene is cloned into the LentiLox3.7 lentivirusvector (as described in Marchetto et al.). The fibroblasts are infectedwith retroviral reprogramming vectors (Sox2, Oct4, c-Myc and Klf4), Twodays after infection, fibroblasts are plated on mitotically inactivatedmouse embryonic fibroblasts with hESC medium. After 2 weeks, iPSCcolonies that emerge from the background of fibroblasts are manuallypicked and transferred to feeder-free conditions on matrigel-coateddishes (BD) using embryonic stem cell culture media mTeSR™ (Stem CellTechnologies) and passaged manually. Gene expression profiles of thegenerated clones are measured using human genome Affymetrix Gene Chip™arrays to confirm that reprogramming is successful.

Neural Differentiation: NPCs and Mature Neurons

To obtain neural progenitor cells (NPCs), embryoid bodies (EBs) areformed by mechanical dissociation of cell clusters and plating ontolow-adherence dishes in hESC medium without FGF2 for 5-7 days. Afterthat, EBs are plated onto poly-ornithine/laminin-coated dishes inDMEM/F12 plus N2 medium (serum-free supplement for growth and expressionof post-mitotic cells). Resulting rosettes are collected after 7 daysand dissociated with accutase and plated onto coated dishes with NPCmedia (DMEM/F12; 0.5×N2; 0.5×B27 and FGF2). Homogeneous populations ofNPCs are achieved after 1-2 passages with accutase in the samecondition. To obtain mature neurons, floating EBs are treated with 1 uMor retinoic acid for 3 weeks (giving the total time of differentiationof 4 weeks). Mature EBs are dissociated with papain and DNAse for 1 h at37° C. and plated in poly-ornithine/laminin-coated dishes in NPC mediawithout FGF2.

Treatment with G-2-MePE

RTT neuronal cultures are treated with G-2-MePE (1 nM-10M) for 1 week,

Immunocytochemistry and Quantification of Neuronal Morphology

Cells are fixed in 4% paraformaldehyde and permeabilized with 0.5%Triton-X100 in PBS. Cells are then blocked in PBS containing 0.5%Triton-X100 and 5% donkey serum for 1 h at room temperature. Fluorescentsignals are detected using a Zeiss inverted microscope and images areprocessed with Photoshop CS3. The following primary antibodies are used:TRA-1-60, TRA-1-81 (1:100), Nanog and Lin28 (1:500), human Nestin(1:100), Tuj-1 (1:500), Map2 (1:100); meCP2 (1:1000; VGLUT1 (1:200),Psd95 (1:500), GFP (1:200), Sox1 (1:250), Mushasil (1:200) and me3H3K27(1:500). Cell soma size is measured using suitable software (e.g.ImageJ) after identification of neurons using the Syn::EGFP™. Themorphologies of neuronal dendrites and spines are studied from anindividual projection of z-stacks optical sections and scanned at 0.5 umincrements that correlate with the resolution valued at z-plane. Eachoptical section is the result of 3 scans at 500 lps followed by Kalmanfiltering. For synapse quantification, images are taken by a z-step of 1um using Biorad radiance 2100™ confocal microscope. Synapsequantification is done blinded to genotype. Only VGLUT1 puncta alongMap2-positive processes are counted. Statistical significance is testedusing 2-way ANOVA test and Bonferroni post-test.

Calcium Imaging

Neuronal networks derived from human iPSCs are infected with thelentiviral vector carrying the Syn:DsRed reporter construct. Cellcultures are washed twice with sterile Krebs HEPES Buffer (KHB) andincubated with 2-5 μM Fluo-4AM™ (Molecular Probes/Invitrogen, Carlsbad,Calif.) in KHB for 40 minutes at room temperature. Excess dye is removedby washing twice with KHB, and an additional 20 minutes incubation isdone to equilibrate intracellular dye concentration and allowde-esterification. Time-lapse image sequences (100× magnification) of5000 frames are acquired at 28 Hz with a region of 336×256 pixels, usinga Hamamatsu ORCA-ER™ digital camera (Hamamatsu Photonics K.K., Japan)with a 488 nm (FITC) filter on an Olympus IX81 inverted fluorescenceconfocal microscope (Olympus Optical, Japan). Images are acquired withMetaMorph 7.7™ (MDS Analytical Technologies, Sunnyvale, Calif.). Imagesare subsequently processed using ImageJ™ and custom written routines inMatlab 7.2™ (Mathworks, Natick, Mass.).

Electrophysiology

Whole-cell patch clamp recordings are performed from cells co-culturedwith astrocytes after 6 weeks of differentiation. The bath is constantlyperfused with fresh HEPES-buffered saline (see supplemental methods forrecipe). The recording micropipettes (tip resistance 3-6 MSG) are filledwith internal solution described in the Supplemental materials.Recordings are made using Axopatch 200B™ amplifier (Axon Instruments).Signals are filtered at 2 kHz and sampled at 5 kHz. The whole-cellcapacitance is fully compensated. The series resistance is uncompensatedbut monitored during the experiment by the amplitude of the capacitivecurrent in response to a 10-mV pulse. All recordings are performed atroom temperature and chemicals are purchased from Sigma. Frequency andamplitude of spontaneous postsynaptic currents are measured with theMini Analysis Program™ software (Synaptosoft, Leonia, N.J.). Statisticalcomparisons of WT and RTT groups are made using the non-parametricKolmogorov-Smirnov two-tailed test, with a significance criterion ofp=0.05. EPSCs are blocked by CNQX or DNQX (10-20 μM) and IPSPs areinhibited by bicuculine (20 μM).

Results

RTT iPSC-derived neurons are characterized by decreased number ofglutamatergic synapses, reduced spine density and smaller soma size. RTTneurons also show certain electophysiological defects, i.e. asignificant decrease in frequency and amplitude of spontaneous synapticcurrents when compared to controls. The RTT neurons show a decreasedfrequency of intracellular calcium transients.

We test G-2-MePE in the above model to test whether any of thepathologies of the RTT phenotype can be attenuated.

Treatment of the cell cultures with each drug concentration improves allof the morphological and physiological parameters of the treated RTTcell cultures in comparison to the non-treated RTT controls.Specifically, we observe a significant increase in glutamatergic synapsenumbers in the G-2-MePE treated RTT cells. All concentrations ofG-2-MePE treatment increase VGLUT1 puncta number in the RTT-derivedneurons. G-2-MePE treatment normalizes the frequency and amplitude ofspontaneous post-synaptic currents as well as the frequency of calciumtransients generated by synaptic activity of the G-2-MePE treated RTTneurons.

In the present in vitro model of human RTT, the iPSCs derived from RTTpatients and neurons differentiated from them are characterized byabnormalities in the MeCP2 expression. As discussed in the detaileddescription of the invention above, the vast majority of RTT cases areassociated with mutations of the MeCP2 gene. Therefore the efficacy ofG-2-MePE in the present in vitro model of human RTT is reasonablypredictive of its efficacy in a human subject suffering from RTT.

Example 15 Effects of G-2-MePE in Human Beings with Rett Syndrome

Methods

Thirty subjects with Rett Syndrome are recruited, Subjects are femaleand aged between 16 and 29 years (Mean=12.1 SD=4.4). All subjects havean IQ<60 and mutations of the MECP2 gene. Subjects also show ether spikeactivity in the EEG or an increase in lower frequency bands of the BEGas detected by Fast Fourier Transform (FFT). Subjects are instructedthat concomitant medications are to be stable for at least six weeksprior to study. Subjects receiving medication to treat signs ofinattention are tested in the morning and instructed to take theirmedication in the afternoon. Subjects with QTc interval >451 msec areexcluded.

The study is a randomized double blind placebo controlled parallel studywith three doses of either placebo, 10 mg/kg T.I.D oral G-2-MePE forfive days, or 30 mg/kg T.I.D. oral G-2-MePE.

Subjects are tested at baseline using the following instruments: TheRett Syndrome Natural History/Clinical Severity Scale, Aberrant BehaviorChecklist Community Edition (ABC), Vinelands, Clinical Global Impressionof Severity (CGI-S) and their carers completed the Caregiver StrainQuestionnaire (CSQ).

Subjects are brought into clinic on an inpatient basis to enable initialbaseline recordings of EEG, ECG and respiratory rate continuously for 24hours using polysomnography technology. Hand movements are also recordedusing the Q-Sensor™. Derived EEG measures include: spikes per unit timein the EEG, overall power of frequency bands of the EEG, QTc and heartrate variability (HRV), and respiratory irregularities.

Adverse events are also recorded using standard safety measures and theSMURF elicitation of adverse events

Statistically, the effect of treatment with G-2-MePE is analysed byconducting a repeated analysis of covariance (ANCOVA) on the effect oftreatment on change from baseline scores.

Results

Treatment with G-2-MePE produces no more adverse events than are presentduring treatment with placebo, with all adverse events being of shortduration and mild severity. No Serious Adverse Events are reported. Noinstances of increases in QTC are reported.

No effects are seen on respiratory rate or heart rate variability.

Treatment with G-2-MePE produces a significant overall reduction ofspikes per unit time in the EEG. Treatment with 30 mg/kg T.I.D. oralG-2-MePE decreases spike activity compared to placebo. This dose ofG-2-MePE also decreases the power of the delta band of the EEG comparedto placebo.

Treatment with G-2-MePE also reduces total hand movements pertwenty-four hour period as counted using the Q-Sensor™ device. Thiseffect is significant for the 30 mg/kg T.I.D. dose compared to placebo.

Treatment with G-2-MePE has no significant effect overall on the RettSyndrome Natural History/Clinical Severity Score. However, 30 mg/kgT.I.D. oral G-2-MePE, compared to placebo, produces significant effectson the following subscales: “Nonverbal Communication at this visit byexam”; “Epilepsy/Seizures at this visit: and “Hand use”.

Conclusions

Treatment with G-2-MePE produces significant improvements in CentralNervous System function in the present study. Despite relatively shortterm treatment, abnormalities in the electrical activity of the brain isreduced, a clear signal of efficacy. This effect is dose dependent, seenafter treatment 30 mg/kg T.I.D. oral G-2-MePE, These effects mirror theimprovements in CNS function seen in the mecp2 knockout transgenic mousemodel of Rett Syndrome after administration of G-2-MePE.

Dose dependent effects are also seen on hand use, as assessed by anobjective counting device and subjective rating. This is of interestbecause purposeless hand wringing is both characteristic to the RettSyndrome clinical phenotype and is unique to this disorder.

The Non-verbal communication rating of the Rett Syndrome NaturalHistory/Clinical Severity Scale is improved by treatment. This measureprimarily assesses eye contact. This raises the prospect that longerterm treatment with G-2-MePE may improve social relatedness in thepopulation.

G-2-MePE is well tolerated in this population. No effects are seen ineither standard measures or areas of specific concern in the patientpopulation, such as QTc interval prolongation or apnea.

Example 16 Effects of G-2MePE on Human Beings with Autism SpectrumDisorders

Methods

To determine whether G-2-MePE can treat symptoms of ASD, we carry out astudy in human beings with ASD. Twenty subjects with an Autism SpectrumDisorder are recruited. Subjects are male and aged between 16 and 65years (Mean=18.1 SD=3.4). All subjects have an IQ >60 and strictDSM-IV-TR diagnosis of Autistic Disorder or Asperger Disorder, Subjectsalso meet criteria for an Autism Spectrum Disorder according the ADI-Rand ADOS-G instruments, and fulfill the proposed DSM-V criteria for andAutism Spectrum Disorder. Subjects are instructed that concomitantmedications are to be stable for at least six weeks prior to study.Subjects receiving medication to treat signs of inattention are testedin the morning and instructed to take their medication in the afternoon.Subjects better treated with atypical anti-psychotic medicationsindicated for autism are excluded. Subjects are screened for knowngenetic disorders including and those with Fragile X Syndrome ortuberous sclerosis excluded. Subjects with uncontrolled epilepsy areexcluded.

The study is a double blind placebo-controlled crossover study withthree phases. Subjects enter each phase of the crossover in a randomizedorder. In the test phases, subjects receive either placebo, 10 mg/kgT.I.D oral G-2-MePE for five days, or 30 mg/kg T.I.D. oral G-2-MePE.Each phase of the crossover is separated by a washout period of fourteendays.

Subjects are tested at baseline using the following instruments:Wechsler IQ, Abberant Behavior Checklist Community Edition (ABC),Vinelands, Yale-Brown Obsessive Compulsive Scale (YBOCS) compulsionsubscale, Social Responsiveness Scale (SRS), Clinical Global Impressionof Severity (CGI-S) and their carers complete the Caregiver StrainQuestionnaire (CSQ).

Subjects are administered two tasks—the Reading the Mind in the EyesTest—Revised (RMET) and an Eye Tracking (ET) task, as well as ClinicalGlobal Impression of Improvement (CGI-1). Tasks commence two hoursfollowing administration of placebo or either dose of G-2-MePE. The RMETis a computer based task that assesses one's ability to read emotionsfrom the eyes of subtle affective facial expressions and is a widelyused test of emotion recognition in patients with autism (2001).Importantly, the RMET is capable of detecting improvement with even asingle dose of a pharmacological agent (Guastella et al., 2010). Eyetracking issues are characteristic of patients with autism who spendless time looking at the eyes of photographs of human faces. Again, asingle administration of a pharmacologic intervention can ameliorate eyetracking deficits in autism (Andari et al, 2010).

Adverse events are also recorded using standard safety measures.

Statistically, the effect of treatment with G-2-MePE is analysed byconducting a repeated analysis of covariance (ANCOVA) on the effect oftreatment on change from baseline scores.

Results

Treatment with G-2-MePE produces no more adverse events than werepresent during treatment with placebo, with all adverse events being ofshort duration and mild severity. No Serious Adverse Events arereported.

Treatment with G-2-MePE produces a significant overall improvement inperformance of the RMET test. Treatment with 30 mg/kg T.I.D. oralG-2-MePE increases the percent correct responses on the RMET.

Treatment with G-2-MePE produces a significant overall improvement intime spent looking at the eye region in the ET test. CGI-1 scores at theend of treatment periods show a significant difference. Positivetreatment effects are correlated with baseline CSQ scores.

Conclusions

Treatment with G-2-MePE produces significant improvements in performancein the Reading the Mind in the Eyes Test—Revised, and in performance ofan Eye Tracking task. This effect is dose dependent, seen aftertreatment 30 mg/kg T.I.D. oral G-2-MePE.

Improvement in these measures is reflective of an improvement inprocessing of social information processing. Social interaction deficitsare a core symptom diagnostic for autism spectrum disorders, and this istherefore a key finding.

G-2-MePE also produces an overall improvement in function as indexed bythe Clinical Global Impression of Improvement. Free text annotation ofthe Case Report Forms from the study indicate this effect related to animprovement in social relatedness. This implies that the changes seen inthe RMET and ET task may have relevance to social activity in dailylife.

G-2-MePE is well tolerated in this population.

Example 17 Animal Models for Determining Effects of G-2-MePE on AutismSpectrum Disorders

Effects of G-2-MePE are further tested in the following genetic modelsof ASD: the Tbx1 heterozygous mouse, the Cntnap2 knockout mouse and theSic9a6 knockout mouse. G-2-MePE is also tested in the fmr1 knockoutmouse model of Fragile X Syndrome.

Tbx1.

Mutations of the TBX1 gene are associated with Autism Spectrum Disorders(Paylor et al., 2006). Transgenic Tbx1 mice are selectively impaired insocial interaction, ultrasonic vocalization, repetitive behaviors andworking memory (Hiramoto et al., 2011).

Cntnap2.

Two-thirds of patients with mutations of the contactin associatedprotein-like 2 (CNTNAP2) gene are diagnosed with an Autism SpectrumDisorder (Alarcon et al., 2008; Arking et al., 2008; Bakkaloglu et al.,2008; Strauss et al., 2006; Vernes et al., 2008). Cntnap2 knockout (KO)mice exhibit ASD-related phenotypes in social behavior, ultrasonicvocalization and repetitive behaviors (Penagarikano et al., 2011).

Slc9a6.

This gene has been implicated in syndromic ASD and encodes thesodim-hydrigen exchanger 6 (NHE6). Mutations in SLC9A6 are associatedwith intellectual disability (Gilfillan et al., 2008) and autisticbehavior (Garbern et al., 2010). On Slc9a6 KO mice exhibit motorhyper-activity and cerebellar dysfunction (Stromme et al., 2011).

Fmr1.

Silencing of the FMR1 gene produces Fragile X Syndrome, the phenotype ofwhich includes autism; two thirds of patients with Fragile X Syndromemeet screening criteria for an Autism Spectrum Disorder (Harris et al.,2008). Pediatric patients with Fragile X Syndrome also show loweredseizure threshold. The fmr1 knockout mouse replicates much of thephenotype of Fragile X Syndrome, including juvenile seizuresusceptibility (Yan at al., 2004).

Methods

Animals in each of the above models are generated in accordance with themethodology described in the cited literature. Wild type equivalents arealso obtained for each genetic model. Animals in each model are dividedinto three groups (n=10 to n=20): placebo treated wild type mice, mutantG-2-MePE-treated group and mutant placebo-treated control group.

The treatments are administered intraperitoneally: placebo (saline) or20 mg/kg/day of G-2-MePE.

Measures of key features of ASD as displayed in each model are taken inaccordance with the cited literature.

Results

G-2-MePE treatment significantly improves all measures associated withthe ASD phenotype.

REFERENCES

The following references and all patents, patent applications and otherpublications cited herein are incorporated fully by reference.

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We claim:
 1. A method for treating a human being suffering from asymptom of Rett Syndrome, comprising oral administration to said humanbeing an effective amount of an aqueous solution ofGlycyl-2-Methyl-L-prolyl-L-glutamate (G-2-MePE).
 2. The method of claim1, said effective amount of G-2-MePE being in the range of about 1 mg/Kgto about 100 mg/Kg.
 3. The method of claim 1, where said G-2-MePE isadministered in a dose of about 60 mg/kg twice per day (“b.i.d.”). 4.The method of claim 1, where said G-2-MePE is administered to a mucosaof said human being.
 5. The method of claim 1, further comprisingadministering to the human being a second therapeutic agent selectedfrom a group consisting of: insulin-like growth factor-I (IGF-I),insulin-like growth factor-II (IGF-II), glycyl-prolyl-glutamate (GPE),transforming growth factor-β1, activin, growth hormone, nerve growthfactor, brain-derived neurotrophic factor (BDNF), growth hormone bindingprotein, IGF-binding proteins (especially IGFBP-3), basic fibroblastgrowth factor, acidic fibroblast growth factor, the hst/Kfgk geneproduct, FGF-3, FGF-4, FGF-6, keratinocyte growth factor,androgen-induced growth factor, int-2, fibroblast growth factorhomologous factor-1 (FHF-1), FHF-2, FHF-3, FHF-4, keratinocyte growthfactor 2, glial-activating factor, FGF-10 and FGF-16, ciliaryneurotrophic factor, brain derived growth factor, neurotrophin 3,neurotrophin 4, bone morphogenetic protein 2 (BMP-2), glial-cell linederived neurotrophic factor, activity-dependant neurotrophic factor,cytokine leukaemia inhibiting factor, oncostatin M, interleukin),α-interferon, β-interferon, γ-, consensus interferon, TNF-α,clomethiazole; kynurenic acid, Semax, tacrolimus,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol,andrenocorticotropin-(4-9), analog [ORG 2766], dizolcipine (MK-801),selegiline; mematine, NPS1506, GV1505260, MK-801, GV150526;2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), LY303070,LY300164; anti-MAdCAM-1mAb, MECA-367 (ATCC accession no. HB-9478),fenobam, fluoxetine, and risperidone.
 6. The method of claim 1, wheresaid dose of said compound is 10 mg/kg three times per day or 30 mg/kgthree times per day.
 7. The method of claim 1, said treatment producingan improvement in said symptom as assessed by one or more behavioraltest selected from the group consisting of The Rett Syndrome NaturalHistory/Clinical Severity Scale, Aberrant Behavior Checklist CommunityEdition (ABC), Vinelands, Clinical Global Impression of Severity (CGI-S)and their carers completed the Caregiver Strain Questionnaire (CSQ), orone or more physiological test selected from the group consisting ofelectroencephalogram (EEG) spike frequency, overall power in frequencybands of an EEG, hand movement, QTc and heart rate variability (HRV),and respiratory irregularities compared to control animals not sufferingfrom said symptom.